Discordant and chameleon sequences: their distribution and implications for amyloidogenicity

Identification of ambiguous encoding in protein secondary structure is paramount to develop an understanding of key protein segments underlying amyloid diseases. We investigate two types of structurally ambivalent peptides, which were hypothesized in the literature as indicators of amyloidogenic proteins: discordant α-helices and chameleon sequences. Chameleon sequences are peptides discovered experimentally in different secondary-structure types. Discordant α-helices are α-helical stretches with strong β-strand propensity or prediction. To assess the distribution of these features in known protein structures, and their potential role in amyloidogenesis, we analyzed the occurrence of discordant α-helices and chameleon sequences in nonredundant sets of protein domains (n = 4263) and amyloidogenic proteins extracted from the literature (n = 77). Discordant α-helices were identified if discordance was observed between known secondary structures and secondary-structure predictions from the GOR-IV and PSIPRED algorithms. Chameleon sequences were extracted by searching for identical sequence words in α-helices and β-strands. We defined frustrated chameleons and very frustrated chameleons based on varying degrees of total β propensity ≥α propensity. To our knowledge, this is the first study to discern statistical relationships between discordance, chameleons, and amyloidogenicity. We observed varying enrichment levels for some categories of discordant and chameleon sequences in amyloidogenic sequences. Chameleon sequences are also significantly enriched in proteins that have discordant helices, indicating a clear link between both phenomena. We identified the first set of discordant-chameleonic protein segments we predict may be involved in amyloidosis. We present a detailed analysis of discordant and chameleons segments in the family of one of the amyloidogenic proteins, the Prion Protein.     

The relationship between amyloid structure and cytotoxicity

Self-assembly of proteins and peptides into amyloid structures has been the subject of intense and focused research due to their association with neurodegenerative, age-related human diseases and transmissible prion diseases in humans and mammals. Of the disease associated amyloid assemblies, a diverse array of species, ranging from small oligomeric assembly intermediates to fibrillar structures, have been shown to have toxic potential. Equally, a range of species formed by the same disease associated amyloid sequences have been found to be relatively benign under comparable monomer equivalent concentrations and conditions. In recent years, an increasing number of functional amyloid systems have also been found. These developments show that not all amyloid structures are generically toxic to cells. Given these observations, it is important to understand why amyloid structures may encode such varied toxic potential despite sharing a common core molecular architecture. Here, we discuss possible links between different aspects of amyloidogenic structures and assembly mechanisms with their varied functional effects. We propose testable hypotheses for the relationship between amyloid structure and its toxic potential in the context of recent reports on amyloid sequence, structure, and toxicity relationships.     

Specific Chaperones and Regulatory Domains in Control of Amyloid Formation

Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology.     

Amyloidogenic determinants are usually not buried

Background  

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

Amphoterin includes a sequence motif which is homologous to the Alzheimer's beta-amyloid peptide (Abeta), forms amyloid fibrils in vitro, and binds avidly to Abeta

Many of the proteins associated with amyloidoses have been found to share structural and sequence similarities, which are believed to be responsible for their capability to form amyloid fibrils. Interestingly, some proteins seem to be able to form amyloid-like fibrils although they are not associated with amyloidoses. This indicates that the ability to form amyloid fibrils may be a general property of a greater number of proteins not associated with these diseases. In the present work, we have searched for amyloidogenic consensus sequences in two current protein/peptide databases and show that many proteins share structures which can be predicted to form amyloid. One of these potentially amyloidogenic proteins is amphoterin (also known as HMG-1), involved in neuronal development and a ligand for the receptor for advanced glycation end products (RAGE). It contains an amyloidogenic peptide fragment which is highly homologous to the Alzheimer's amyloid beta-peptide. If enzymatically released from the native protein, it forms amyloid-like fibrils which are visible in electron microscopy, exhibit apple green birefringence under polarized light after Congo red staining, and increases thioflavin T fluorescence. This fragment also shows high affinity to Abeta as a free peptide or while part of the native protein. Our results support the hypothesis that the potential to form amyloid is a common characteristic of a number of proteins, independent of their relation to amyloidoses, and that this potential can be predicted based on the physicochemical properties of these proteins.     

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.     

Binding of the dye congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences.

The three-dimensional structure has been determined of a complex of the dye Congo Red, a specific stain for amyloid deposits, bound to the amyloid protein insulin. One dye molecule intercalates between two globular insulin molecules at an interface formed by a pair of anti-parallel beta-strands. This result, together with analysis of the primary sequences of other amyloidogenic proteins and peptides suggests that this mode of dye-binding to amyloid could be general. Moreover, the structure of this dye-binding interface between protein molecules provides an insight into the polymerization of amyloidogenic proteins into amyloid fibres. Thus the detailed characterization, at a resolution of 2.5 A, of the dye binding site in insulin could form a basis for the design of agents targeted against a variety of amyloid deposits.     

Nonpolar substitution at the C-terminus of the prion protein, a mimic of the glycosylphosphatidylinositol anchor, partially impairs amyloid fibril formation

In contrast to most amyloidogenic proteins or peptides that do not contain any significant posttranslational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant nonpeptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with an N-myristoylamidomaleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) it substantially decreased the extent of fibrillation, presumably due to off-pathway aggregation, and (ii) it prohibited assembly of filaments into higher order fibrils by preventing their lateral association. The negative effect on lateral association was abolished if the myristoylated moiety at the C-terminus was replaced by a polar group of similar size or by a hydrophobic group of smaller size. When preformed PrP fibrils were provided as seeds, myristoylated PrP supported fibril elongation and formation of higher order fibrils composed of several filaments. Our studies illustrate that, despite a bulky hydrophobic moiety at C-terminus, myristoylated PrP can still incorporate into fibrillar structure and that the C-terminal hydrophobic substitution does not affect the size of the proteinase K resistant core but controls the mode of lateral assembly of filaments into higher order fibrils.     

Structural trees for protein superfamilies

Structural trees for large protein superfamilies, such as β proteins with the aligned β sheet packing, β proteins with the orthogonal packing of α helices, two-layer and three-layer α/β proteins, have been constructed. The structural motifs having unique overall folds and a unique handedness are taken as root structures of the trees. The larger protein structures of each superfamily are obtained by a stepwise addition of α helices and/or β strands to the corresponding root motif, taking into account a restricted set of rules inferred from known principles of the protein structure. Among these rules, prohibition of crossing connections, attention to handedness and compactness, and a requirement for α helices to be packed in α-helical layers and β strands in β layers are the most important. Proteins and domains whose structures can be obtained by stepwise addition of α helices and/or β strands to the same root motif can be grouped into one structural class or a superfamily. Proteins and domains found within branches of a structural tree can be grouped into subclasses or subfamilies. Levels of structural similarity between different proteins can easily be observed by visual inspection. Within one branch, protein structures having a higher position in the tree include the structures located lower. Proteins and domains of different branches have the structure located in the branching point as the common fold. Proteins 28:241–260, 1997. © 1997 Wiley-Liss Inc.     

Exogenous amyloidogenic proteins function as seeds in amyloid β-protein aggregation

Amyloid β-protein (Aβ) aggregation is considered to be a critical step in the neurodegeneration of Alzheimer's disease (AD). In addition to Aβ, many proteins aggregate into the amyloid state, in which they form elongated fibers with spines comprising stranded β-sheets. However, the cross-seeding effects of other protein aggregates on Aβ aggregation pathways are not completely clear. To investigate the cross-seeding effects of exogenous and human non-CNS amyloidogenic proteins on Aβ aggregation pathways, we examined whether and how sonicated fibrils of casein, fibroin, sericin, actin, and islet amyloid polypeptide affected Aβ40 and Aβ42 aggregation pathways using the thioflavin T assay and electron microscopy. Interestingly, the fibrillar seeds of all amyloidogenic proteins functioned as seeds. The cross-seeding effect of actin was stronger but that of fibroin was weaker than that of other proteins. Furthermore, our nuclear magnetic resonance spectroscopic studies identified the binding sites of Aβ with the amyloidogenic proteins. Our results indicate that the amyloidogenic proteins, including those contained in foods and cosmetics, contribute to Aβ aggregation by binding to Aβ, suggesting their possible roles in the propagation of Aβ amyloidosis.     

蛋白质错误折叠相关疾病的多肽药物研究进展

蛋白质和多肽发生错误折叠形成不可溶的淀粉样纤维的过程,与阿尔茨海默病、帕金森病等多种神经退行性疾病密切相关。这些疾病可导致认知能力下降以及运动缺陷等症状。虽然已有多种相关治疗方案处于临床试验中,但目前仍无明确有效的方法可治愈或长期减缓疾病的进展。探寻和研究抑制淀粉样聚集、识别并促进毒性聚集物清除的抑制剂分子是药物研发的重要策略之一。在不同类型的抑制剂中,多肽类抑制剂因具有高特异性、低毒性、多样性,以及修饰后的抗水解稳定性和血脑屏障通透性,有望成为候选药物分子。本文总结了针对阿尔茨海默病相关的Aβ和Tau蛋白以及帕金森病相关的α-synuclein蛋白淀粉样纤维化的多肽抑制剂研究进展。基于淀粉样纤维化核心序列及纤维核心结构进行合理设计,或通过随机筛选,均可获得多肽抑制剂。这些天然和非天然的多肽分子大多具有抑制淀粉样纤维化、解聚成熟纤维和降低细胞毒性的作用,其中一些多肽在退行性疾病动物模型实验中,显示出降低脑损伤和缓解认知及运动障碍的效果。这些研究揭示了多肽作为蛋白质错误折叠和聚集相关疾病药物的特点,为研发一类新的有效药物奠定了基础。     

Spectroscopy-Based Modelling of the 3D Structure of the β Subunit of the High Affinity IgE Receptor

Abstract

The high affinity IgE receptor, possesses a tetrameric structure. The 243 residue β subunit is a polytopic protein with four hydrophobic membrane-spanning segments, whereas the individual α and γ subunits are bitopic proteins each containing one transmembrane domain in their monomeric form. In the proposed topographical model (Blank et al., 1989), the four trans-membrane α helices of the β subunit are connected by three loop sequences.

To study the individual subunits and intact receptor, this membrane protein was divided into domains such as its loop peptides, cytoplasmic peptides and transmembrane helices according to Blank et al., 1989. The 3D structure of the synthesized loop peptides and cytoplasmic peptides were calculated; CD and/or NMR data were used as appropriate to generate the resultant structures which were then used as data basis for the higher level calculations.

The four individual transmembrane helices of the β subunit were characterised, first of all, by mapping the relative lipophilicity of their surfaces using lipophilic probes. A second procedure, docking of the individual helices in pairs, was used to predict helix–helix interactions.

The data on the relative lipophilicity of the surfaces as well as the surfaces that favoured helix–helix interactions were used in combination with the spectroscopy-based structures of the loops and cytoplasmic domains to calculate via molecular dynamics, the helix arrangement and 3D structure of the β subunit of the high affinity IgE receptor. In the final analysis, the molecular simulations yielded two structures of the β subunit, which should form a basis for the modelling of the whole high affinity IgE receptor.     

Porins and Amyloids are Coded by Similar Sequence Motifs

The relationship between amino acid sequences of the β‐hairpin structures and amyloidogenic β‐arcade‐forming motifs are of special interest because, similar to amyloid fibrils, most of the β‐hairpin repeat (BHR) structures have the so‐called cross‐β arrangement. Moreover, β‐hairpin is considered as a probable intermediate structure in amyloidogenesis. In this work, a bioinformatics sequence analysis of the known BHR structures is performed in search of amylodogenic motifs able to form β‐arcade fibrils. The analysis shows that the occurrence of the predicted β‐arcade motifs in the BHR regions is very different depending on the BHR structural fold, cellular localization, and phylogeny. One of the most striking observations is the high level of sequence similarity between the BHRs of membranous porins and β‐arcade motifs. This sequence similarity provides additional evidence that the structure of the membranous porins and annular amyloid oligomers may bear a resemblance. Moreover, these results explain how some amyloidogenic sequence can fold in either the ring‐like shape oligomers or elongated amyloid fibrils. It has been also found that potentially lethal amyloidogenic β‐arcade motifs are absent in the elongated BHR structures of intracellular eukaryotic proteins. It allows to hypothesize that, in this case, the selective evolutionary pressure acts against aggregation.     

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.     

Insights into Stabilizing Forces in Amyloid Fibrils of Differing Sizes from Polarizable Molecular Dynamics Simulations

Pathological aggregation of amyloid-forming proteins is a hallmark of a number of human diseases, including Alzheimer's, type 2 diabetes, Parkinson's, and more. Despite having very different primary amino acid sequences, these amyloid proteins form similar supramolecular, fibril structures that are highly resilient to physical and chemical denaturation. To better understand the structural stability of disease-related amyloids and to gain a greater understanding of factors that stabilize functional amyloid assemblies, insights into tertiary and quaternary interactions are needed. We performed molecular dynamics simulations on human tau, amyloid-β, and islet amyloid polypeptide fibrils to determine key physicochemical properties that give rise to their unique characteristics and fibril structures. These simulations are the first of their kind in employing a polarizable force field to explore properties of local electric fields on dipole properties and other electrostatic forces that contribute to amyloid stability. Across these different amyloid fibrils, we focused on how the underlying forces stabilize fibrils to elucidate the driving forces behind the protein aggregation. The polarizable model allows for an investigation of how side-chain dipole moments, properties of structured water molecules in the fibril core, and the local environment around salt bridges contribute to the formation of interfaces essential for fibril stability. By systematically studying three amyloidogenic proteins of various fibril sizes for key structural properties and stabilizing forces, we shed light on properties of amyloid structures related to both diseased and functional states at the atomistic level.     

Fibrillation of α‐lactalbumin: Effect of crocin and safranal,two natural small molecules from Crocus sativus

Formation of toxic amyloid structures is believed to be associated with various late‐onset neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The fact that many proteins in addition to those that are associated with clinical conditions have the potential to form amyloid fibrils in vitro provides opportunities for studying the fundamentals of protein aggregation and amyloid formation in model systems. Accordingly, considerable interest and effort has been directed toward developing small molecules to inhibit the formation of fibrillar assemblies and their associated toxicities. In the present study, we investigated the inhibitory effect of crocin and safranal, two principal components of saffron, on fibrillation of apo‐α‐lactalbumin (a‐α‐LA), used as a model protein, under amyloidogenic conditions. In the absence of any ligand, formation of soluble oligomers became evident after 18 h of incubation, followed by subsequent appearance of mature fibrils. Upon incubation with crocin or safranal, while transition phase to monomeric beta structures was not significantly affected, formation of soluble oligomers and following fibrillar assemblies were inhibited. While both safranal and crocin had the ability to bind to hydrophobic patches provided in the intermediate structures, and thereby inhibit protein aggregation, crocin was found more effective, possibly due to its simultaneous hydrophobic and hydrophilic character. Cell viability assay indicated that crocin could diminish toxicity while safranal act in reverse order. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 854–865, 2010.     

On the significance of alternating patterns of polar and non-polar residues in beta-strands

A common assumption about protein sequences in beta-strands is that they have alternating patterns of polar and non-polar residues. It is thought that such patterns reflect the interior/exterior geometry of amino acid residue side-chains on a beta-sheet. Here we study the prevalence of simple hydrophobicity patterns in parallel and antiparallel beta-sheets in proteins of known structure and in the sequences of amyloidogenic proteins. The occurrence of 32 possible pentapeptide binary patterns (polar (P)/non-polar (N)) is computed in 1911 non-homologous protein structures. Despite their tendency to aggregate in experimentally designed proteins, the purely alternating hydrophobic/polar patterns (PNPNP and NPNPN) are most frequent in beta-sheets, typically occurring in antiparallel strands. The overall distribution of the pentapeptide binary patterns is significantly different in strands within parallel and antiparallel sheets. In both types of sheets, complementary patterns (where the hydrophobic and polar residues pair with one another) associate preferentially. We do not find alternating patterns to be common in amyloidogenic proteins or in short fragments involved directly in amyloid formation. However, we do note some similarities between patterns present in amyloidogenic sequences and those in parallel strands.     

Correlation between the structural stability and aggregation propensity of proteins

Protein aggregation, being an outcome of improper protein folding, is largely dependent on the folding kinetics of a protein. Previous studies have reported a positive correlation between the stability of the secondary structural elements of a protein and their rate of folding/unfolding. In this in silico study, the secondary and tertiary structures of proteins a) that form inclusion bodies on overexpression in Escherichia coli, b) that form amyloid fibrils and c) that are soluble on overexpression in E. coli are analyzed for certain features that are known to be associated with structural stability. The study revealed that the soluble proteins seem to have a higher rate of folding (based on contact order) and a lower percentage of exposed hydrophobic residues as compared to the inclusion body forming or amyloidogenic proteins. The soluble proteins also seem to have a more favored helix and strand composition (based on the known secondary structural propensities of amino acids). The secondary structure analyses also reveal that the evolutionary pressure is directed against protein aggregation. This understanding of the positive correlation between structural stability and solubility, along with the other parameters known to influence aggregation, could be exploited in the design of mutations aimed at reducing the aggregation propensity of the proteins.     

Promotion of amyloid beta protein misfolding and fibrillogenesis by a lipid oxidation product

Oxidatively damaged lipid membranes are known to promote the aggregation of amyloid β proteins and fibril formation. Oxidative damage typically produces 4-hydroxy-2-nonenal when lipid membranes contain ω-6 polyunsaturated fatty acyl chains, and this compound is known to modify the three His residues in Aβ proteins by Michael addition. In this report, the ability of 4-hydroxy-2-nonenal to reproduce the previously observed amyloidogenic effects of oxidative lipid damage on amyloid β proteins is demonstrated and the mechanism by which it exerts these effects is examined. Results indicate that 4-hydroxy-2-nonenal modifies the three His residues in amyloid beta proteins, which increases their membrane affinity and causes them to adopt a conformation on membranes that is similar to their conformation in a mature amyloid fibril. As a consequence, fibril formation is accelerated at relatively low protein concentrations, and the ability to seed the formation of fibrils by unmodified amyloid beta proteins is enhanced. These in vitro findings linking oxidative stress to amyloid fibril formation may be significant to the in vivo mechanism by which oxidative stress is linked to the formation of amyloid plaques in Alzheimer's disease.     

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.