Is it possible to predict amyloidogenic regions from sequence alone?

Identification of potentially amyloidogenic regions in polypeptide chains is very important because the amyloid fibril formation can be induced in most normal proteins. In our work we suggest a new method to detect amyloidogenic regions in protein sequence. It is based on the assumption that packing is tight inside an amyloid and therefore regions which could potentially pack well would have a tendency to form amyloids. This means that the regions with strong expected packing of residues would be responsible for the amyloid formation. We use this property to identify potentially amyloidogenic regions in proteins basing on their amino acid sequences only. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Predictions of the regions which are responsible for the formation of amyloid fibrils in proteins unrelated to disease have been also done.     

Prediction of amyloid aggregation in vivo

Many human diseases owe their pathology, to some degree, to the erroneous conversion of proteins from their soluble state into fibrillar, β-structured aggregates, often referred to as amyloid fibrils. Neurodegenerative diseases, such as Alzheimer and spongiform encephalopathies, as well as type 2 diabetes and both localized and systemic amyloidosis, are among the conditions that are associated with the formation of amyloid fibrils. Several mathematical tools can rationalize and even predict important parameters of amyloid fibril formation. It is not clear, however, whether such algorithms have predictive powers for in vivo systems, in which protein aggregation is affected by the presence of other biological factors. In this review, we briefly describe the existing algorithms and use them to predict the effects of mutations on the aggregation of specific proteins, for which in vivo experimental data are available. The comparison between the theoretical predictions and the experimental data obtained in vivo is shown for each algorithm and experimental data set, and statistically significant correlations are found in most cases.     

Stem-forming regions that are essential for the amyloidogenesis of prion proteins

Prion diseases represent fatal neurodegenerative disorders caused by the aggregation of prion proteins. With regard to the formation of the amyloidogenic cross-β-structure, the initial mechanism in the conversion to a β-structure is critically important. To explore the core regions forming a stem of the amyloid, we designed and prepared a series of peptides comprised of two native sequences linked by a turn-inducing dipeptide moiety and examined their ability to produce amyloids. A sequence alignment of the peptides bearing the ability to form amyloid structures revealed that paired strands consisting of VNITI (residues 180-184) and VTTTT (residues 189-193) are the core regions responsible for initiating the formation of cross-β-structures and for further ordered aggregation. In addition, most of the causative mutations responsible for inherited prion diseases were found to be located in these stem-forming regions on helix H2 and their counterpart on helix H3. Moreover, the volume effect of the nonstem domain, which contains ~200 residues, was deduced to be a determinant of the nature of the association such as oligomerization, because the stem-forming domain is only a small part of a prion protein. Taken together, we conclude that the mechanism underlying the initial stage of amyloidogenesis is the exposure of a newly formed intramolecular β-sheet to a solvent through the partial transition of a native structure from an α-helix to a β-structure. Our results also demonstrate that prion diseases caused by major prion proteins except the prions of some fungi such as yeast are inherent only in mammals, as evidenced by a comparison of the corresponding sequences to the stem-forming regions among different animals.     

Detecting hidden sequence propensity for amyloid fibril formation

The preponderance of evidence implicates protein misfolding in many unrelated human diseases. In all cases, normal correctly folded proteins transform from their proper native structure into an abnormal beta-rich structure known as amyloid fibril. Here we introduce a computational algorithm to detect nonnative (hidden) sequence propensity for amyloid fibril formation. Analyzing sequence-structure relationships in terms of tertiary contact (TC), we find that the hidden beta-strand propensity of a query local sequence can be quantitatively estimated from the secondary structure preferences of template sequences of known secondary structure found in regions of high TC. The present method correctly pinpoints the minimal peptide fragment shown experimentally as the likely local mediator of amyloid fibril formation in beta-amyloid peptide, islet amyloid polypeptide (hIAPP), alpha-synuclein, and human acetylcholinesterase (AChE). It also found previously unrecognized beta-strand propensities in the prototypical helical protein myoglobin that has been reported as amyloidogenic. Analysis of 2358 nonhomologous protein domains provides compelling evidence that most proteins contain sequences with significant hidden beta-strand propensity. The present method may find utility in many medically relevant applications, such as the engineering of protein sequences and the discovery of therapeutic agents that specifically target these sequences for the prevention and treatment of amyloid diseases.     

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.     

Linking amyloid protein aggregation and yeast survival

Protein aggregation and amyloid formation lie behind an increasing number of human diseases. Here we describe the application of an "aggregation reporter", in which the test protein is fused to dihydrofolate reductase, as a general method to assess the intracellular solubility of amyloid proteins in eukaryotic background. Because the aggregation state of the target protein is linked directly to yeast cells survival in the presence of methotrexate, protein solubility can be monitored in vivo without the requirement of a functional assay for the protein of interest. In addition, the approach allows the in vivo visualization of the cellular location and aggregated state of the target protein. To demonstrate the applicability of the assay in the screening of genes or compounds that modulate amyloid protein aggregation in living cells, we have used as models the Alzheimer's amyloid β peptide, polyglutamine expansions of huntingtin, α-synuclein and non-aggregating variants thereof. Moreover, the anti-aggregational properties of small molecules and the effects of the yeast protein quality control machinery have also been evaluated using this method.     

The effects of the flavonoid baicalein and osmolytes on the Mg 2+ accelerated aggregation/fibrillation of carboxymethylated bovine 1SS-alpha-lactalbumin

Many protein conformational diseases arise when proteins form alternative stable conformations, resulting in aggregation and accumulation of the protein as fibrillar deposits, or amyloids. Interestingly, numerous proteins implicated in amyloid protein formation show similar structural and functional properties. Given this similarity, we tested the notion that carboxymethylated bovine alpha-lactalbumin (1SS-alpha-lac) could serve as a general amyloid fibrillation/aggregation model system. Like most amyloid forming systems, Mg2+ ions accelerate 1SS-alpha-lac amyloid fibril formation. While osmolytes such as trimethylamine N-oxide (TMAO), and sucrose enhanced thioflavin T detected aggregation, a mixture of trehalose and TMAO substantially inhibited aggregation. Most importantly however, the flavonoid, baicalein, known to inhibit alpha-synuclein amyloid fibril formation, also inhibits 1SS-alpha-lac amyloid with the same apparent efficacy. These data suggest that the easily obtainable 1SS-alpha-lac protein can serve as a general amyloid model and that some small molecule amyloid inhibitors may function successfully with many different amyloid systems.     

Aggregation prone regions in human proteome: Insights from large‐scale data analyses

Protein aggregation leads to several burdensome human maladies, but a molecular level understanding of how human proteome has tackled the threat of aggregation is currently lacking. In this work, we survey the human proteome for incidence of aggregation prone regions (APRs), by using sequences of experimentally validated amyloid‐fibril forming peptides and via computational predictions. While approximately 30 human proteins are currently known to be amyloidogenic, we found that 260 proteins (～1% of human proteome) contain at least one experimentally validated amyloid‐fibril forming segment. Computer predictions suggest that more than 80% of the human proteins contain at least one potential APR and approximately two‐thirds (65%) contain two or more APRs; spanning 3–5% of their sequences. Sequence randomizations show that this apparently high incidence of APRs has been actually significantly reduced by unique amino acid composition and sequence patterning of human proteins. The human proteome has utilized a wide repertoire of sequence‐structural optimization strategies, most of them already known, to minimize deleterious consequences due to the presence of APRs while simultaneously taking advantage of their order promoting properties. This survey also found that APRs tend to be located near the active and ligand binding sites in human proteins, but not near the post translational modification sites. The APRs in human proteins are also preferentially found at heterotypic interfaces rather than homotypic ones. Interestingly, this survey reveals that APRs play multiple, often opposing, roles in the human protein sequence‐structure‐function relationships. Insights gained from this work have several interesting implications towards novel drug discovery and development. Proteins 2017; 85:1099–1118. © 2017 Wiley Periodicals, Inc.     

Prediction of amyloidogenic and disordered regions in protein chains

The determination of factors that influence protein conformational changes is very important for the identification of potentially amyloidogenic and disordered regions in polypeptide chains. In our work we introduce a new parameter, mean packing density, to detect both amyloidogenic and disordered regions in a protein sequence. It has been shown that regions with strong expected packing density are responsible for amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for eight of 12 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Our findings support the concept that the mechanism of amyloid fibril formation is similar for different peptides and proteins. Moreover, we have demonstrated that regions with weak expected packing density are responsible for the appearance of disordered regions. Our method has been tested on datasets of globular proteins and long disordered protein segments, and it shows improved performance over other widely used methods. Thus, we demonstrate that the expected packing density is a useful value with which one can predict both intrinsically disordered and amyloidogenic regions of a protein based on sequence alone. Our results are important for understanding the structural characteristics of protein folding and misfolding.     

Protein particulates: another generic form of protein aggregation?

Protein aggregation is a problem with a multitude of consequences, ranging from affecting protein expression to its implication in many diseases. Of recent interest is the specific form of aggregation leading to the formation of amyloid fibrils, structures associated with diseases such as Alzheimer's disease. The ability to form amyloid fibrils is now regarded as a property generic to all polypeptide chains. Here we show that around the isoelectric point a different generic form of aggregation can also occur by studying seven widely different, nonrelated proteins that are also all known to form amyloid fibrils. Under these conditions gels consisting of relatively monodisperse spherical particulates are formed. Although these gels have been described before for beta-lactoglobulin, our results suggest that the formation of particulates in the regime where charge on the molecules is minimal is a common property of all proteins. Because the proteins used here also form amyloid fibrils, we further propose that protein misfolding into clearly defined aggregates is a generic process whose outcome depends solely on the general properties of the state the protein is in when aggregation occurs, rather than the specific amino acid sequence. Thus under conditions of high net charge, amyloid fibrils form, whereas under conditions of low net charge, particulates form. This observation furthermore suggests that the rules of soft matter physics apply to these systems.     

Amyloid cores in prion domains: Key regulators for prion conformational conversion

Despite the significant efforts devoted to decipher the particular protein features that encode for a prion or prion-like behavior, they are still poorly understood. The well-characterized yeast prions constitute an ideal model system to address this question, because, in these proteins, the prion activity can be univocally assigned to a specific region of their sequence, known as the prion forming domain (PFD). These PFDs are intrinsically disordered, relatively long and, in many cases, of low complexity, being enriched in glutamine/asparagine residues. Computational analyses have identified a significant number of proteins having similar domains in the human proteome. The compositional bias of these regions plays an important role in the transition of the prions to the amyloid state. However, it is difficult to explain how composition alone can account for the formation of specific contacts that position correctly PFDs and provide the enthalpic force to compensate for the large entropic cost of immobilizing these domains in the initial assemblies. We have hypothesized that short, sequence-specific, amyloid cores embedded in PFDs can perform these functions and, accordingly, act as preferential nucleation centers in both spontaneous and seeded aggregation. We have shown that the implementation of this concept in a prediction algorithm allows to score the prion propensities of putative PFDs with high accuracy. Recently, we have provided experimental evidence for the existence of such amyloid cores in the PFDs of Sup35, Ure2, Swi1, and Mot3 yeast prions. The fibrils formed by these short stretches may recognize and promote the aggregation of the complete proteins inside cells, being thus a promising tool for targeted protein inactivation.     

与人类疾病相关的淀粉样短肽

有些天然蛋白质可通过错误折叠形成淀粉样纤维,并进一步沉积导致淀粉样病变,被认为是许多重大人类疾病的病理基础。因此,阐明天然蛋白质错误折叠、聚集形成淀粉样纤维的分子机制,是预防、诊断和治疗相关疾病的关键。研究者们从天然蛋白质中鉴定出许多能够形成淀粉样纤维的关键短肽片段,即淀粉样短肽,对它们形成淀粉样纤维的能力及其在完整蛋白质聚集过程中的决定性作用进行深入研究。本文对近年来人类疾病相关淀粉样短肽的研究展开综述。首先,介绍鉴定淀粉样短肽的标准及其相应的研究方法和技术手段;并回顾近年来与一些重大人类疾病相关的淀粉样短肽,尤其是与神经退行性疾病相关淀粉样短肽的进展情况,对淀粉样短肽中出现频率较高的氨基酸残基及其可能的自组装原理进行总结分析;最后,展望这些淀粉样短肽作为靶点在相关疾病诊断和治疗方面的意义,并初步探讨它们作为新型生物材料在生物医学工程领域的应用前景。本文一方面为阐明天然蛋白质形成淀粉样沉淀的分子机制提供参考,另一方面也为相关疾病的治疗提供思路,同时也为新型生物材料的开发提出潜在的可能性。     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

Towards Multiparametric Fluorescent Imaging of Amyloid Formation: Studies of a YFP Model of α-Synuclein Aggregation

Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein α-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.     

Amyloidogenicity of naturally occurring full‐length animal IAPP variants

The aggregation of the 37‐amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic β‐islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on‐path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full‐length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full‐length IAPP.     

Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains

Protein aggregation is associated with a variety of pathological conditions, including Alzheimer's and Creutzfeldt-Jakob diseases and type II diabetes. Such degenerative disorders result from the conversion of the normal soluble state of specific proteins into aggregated states that can ultimately form the characteristic amyloid fibrils found in diseased tissue. Under appropriate conditions it appears that many, perhaps all, proteins can be converted in vitro into amyloid fibrils. The aggregation propensities of different polypeptide chains have, however, been observed to vary substantially. Here, we describe an approach that uses the knowledge of the amino acid sequence and of the experimental conditions to reproduce, with a correlation coefficient of 0.92 and over five orders of magnitude, the in vitro aggregation rates of a wide range of unstructured peptides and proteins. These results indicate that the formation of protein aggregates can be rationalised to a considerable extent in terms of simple physico-chemical parameters that describe the properties of polypeptide chains and their environment.     

Domain interactions direct misfolding and amyloid formation of yeast phosphoglycerate kinase

There are proteins that are built of two structural domains and are deposited full-length in amyloid plaques formed in various diseases. In spite of the known differences in the mechanisms of folding of single- and multidomain proteins, no published studies can be found that address the role of the domain-domain interactions during misfolding and amyloid formation. By the discovery of the role of domain-domain interactions, here we provide important insight in the submolecular mechanism of amyloid formation. A model system based on yeast phosphoglycerate kinase was designed. This system includes the wild-type yeast phosphoglycerate kinase and single-tryptophan mutants of the individual N and C terminal domains and the complete protein. Electron microscopic measurements proved that amyloid fibrils grow from all mutants under identical conditions as for the wild-type protein. Misfolding and amyloid formation was followed in stopped-flow and manual mixing experiments on the 1 ms to 4 days timescale. Tryptophan fluorescence was used for selective detection of conformational changes accompanying the formation of the amyloidogenic intermediates and the growth of amyloid fibrils. The interactions between the polypeptide chains of the two domains direct the misfolding process from the early steps to the amyloid formation, and influence the final structure. The kinetics of misfolding is different for the individual domains, pointing to the significance of the amino acid sequence. Misfolding of the domains within the complete protein is synchronized indicating that domain-domain interactions direct the misfolding and amyloid formation mechanism.     

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.     

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.     

Inhibition of amyloid formation

Amyloid is aggregated protein in the form of insoluble fibrils. Amyloid deposition in human tissue-amyloidosis-is associated with a number of diseases including all common dementias and type II diabetes. Considerable progress has been made to understand the mechanisms leading to amyloid formation. It is, however, not yet clear by which mechanisms amyloid and protein aggregates formed on the path to amyloid are cytotoxic. Strategies to prevent protein aggregation and amyloid formation are nevertheless, in many cases, promising and even successful. This review covers research on intervention of amyloidosis and highlights several examples of how inhibition of protein aggregation and amyloid formation has been achieved in practice. For instance, rational design can provide drugs that stabilize a native folded state of a protein, protein engineering can provide new binding proteins that sequester monomeric peptides from aggregation, small molecules and peptides can be designed to block aggregation or direct it into non-cytotoxic paths, and monoclonal antibodies have been developed for therapies towards neurodegenerative diseases based on inhibition of amyloid formation and clearance.