Amyloid-like Peptides Research Articles

Data-mining unveils structure–property–activity correlation of viral infectivity enhancing self-assembling peptides

Gene therapy via retroviral vectors holds great promise for treating a variety of serious diseases. It requires the use of additives to boost infectivity. Amyloid-like peptide nanofibers (PNFs) were shown to efficiently enhance retroviral gene transfer. However, the underlying mode of action of these peptides remains largely unknown. Data-mining is an efficient method to systematically study structure–function relationship and unveil patterns in a database. This data-mining study elucidates the multi-scale structure–property–activity relationship of transduction enhancing peptides for retroviral gene transfer. In contrast to previous reports, we find that not the amyloid fibrils themselves, but rather µm-sized β-sheet rich aggregates enhance infectivity. Specifically, microscopic aggregation of β-sheet rich amyloid structures with a hydrophobic surface pattern and positive surface charge are identified as key material properties. We validate the reliability of the amphiphilic sequence pattern and the general applicability of the key properties by rationally creating new active sequences and identifying short amyloidal peptides from various pathogenic and functional origin. Data-mining—even for small datasets—enables the development of new efficient retroviral transduction enhancers and provides important insights into the diverse bioactivity of the functional material class of amyloids.

Acetylenic Bond Layout Drives the Directional Assembly of Amyloid-like Peptides on Graphyne: Implications for Biomedical Safety Applications of Graphyne

Acetylenic Bond Layout Drives the Directional Assembly of Amyloid-like Peptides on Graphyne: Implications for Biomedical Safety Applications of Graphyne

Controllable membrane damage by tunable peptide aggregation with albumin.

Aggregation of otherwise soluble proteins into amyloid structures is a hallmark of many disorders, such as Alzheimer's and Parkinson's disease. There is an increasing evidence that the small aggregations, instead of ordered fibrillar aggregates, are the main structures causing toxicity. However, the studies on the small aggregation phase are limited due to the variety of structures and the complexity of the physiological environment. Here, we showed an engineered co-assembling oppositely charged amyloid-like peptide pair ([II]) as a simple tool to establish methodologies to study the mechanism and kinetics of aggregation and relate its aggregation to toxicity. The toxicity mechanism of [II] is through cell membrane damage and stress, shown with YAP and eIF2α, as in the amyloid protein-initiated diseases. Albumin is demonstrated as an extrinsic and physiologically relevant molecule in controlling the aggregation lag time and toxicity of [II]. This study represents a molecular engineering strategy to create simplistic molecular tools for establishing methodologies to study the aggregation process and kinetics of amyloid-like proteins in various conditions. Understanding the nature of protein aggregation kinetics and linking them to their biological functions through engineered peptides paves the way for future designs and drug development applications.

Orange G is a potential inhibitor of human insulin amyloid fibrillation and can be used as a probe to study mechanism of amyloid fibrillation and its inhibition

The extracellular insoluble deposits of highly ordered cross-β-structure-containing amyloid fibrils form the pathological basis for protein misfolding diseases. As amyloid fibrils are cytotoxic, inhibition of the process is a therapeutic strategy. Several small molecules have been identified and used as fibrillation inhibitors in the recent past. In this work, we investigate the effect of Orange G on insulin amyloid formation using fluorescence-based assays and negative-stain electron microscopy (EM). We show that Orange G effectively attenuates nucleation, thereby inhibiting amyloid fibrillation in a dose-dependent manner. Fluorescence quenching titrations of Orange G showed a reasonably strong binding affinity to native insulin. Binding isotherm measurements revealed the binding of Orange G to pre-formed insulin fibrils too, indicating that Orange G likely binds and stabilizes the mature fibrils and prevents the release of toxic oligomers which could be potential nuclei or templates for further fibrillation. Molecular docking of Orange G with native insulin and amyloid-like peptide structures were also carried out to analyse the contributing interactions and binding free energy. The findings of our study emphasize the use of Orange G as a molecular probe to identify and design inhibitors of amyloid fibrillation and to investigate the structural and toxic mechanisms underlying amyloid formation.

Engineering Nanointerfaces of Au25 Clusters for Chaperone-Mediated Peptide Amyloidosis.

Synthetic nanomaterials possessing biomolecular-chaperone functions are good candidates for modulating physicochemical interactions in many bioapplications. Despite extensive research, no general principle to engineer nanomaterial surfaces is available to precisely manipulate biomolecular conformations and behaviors, greatly limiting attempts to develop high-performance nanochaperone materials. Here, we demonstrate that, by quantifying the length (-SCxR±, x = 3-11) and charges (R- = -COO-, R+ = -NH3+) of ligands on Au25 gold nanochaperones (AuNCs), simulating binding sites and affinities of amyloid-like peptides with AuNCs, and probing peptide folding and fibrillation in the presence of AuNCs, it is possible to precisely manipulate the peptides' conformations and, thus, their amyloidosis via customizing AuNCs nanointerfaces. We show that intermediate-length liganded AuNCs with a specific charge chaperone peptides' native conformations and thus inhibit their fibrillation, while other types of AuNCs destabilize peptides and promote their fibrillation. We offer a microscopic molecular insight into peptide identity on AuNCs and provide a guideline in customizing nanochaperones via manipulating their nanointerfaces.

Solid-state optical properties of self-assembling amyloid-like peptides with different charged states at the terminal ends

The self-assembling of small peptides not only leads to the formation of intriguing nanoarchitectures, but also generates materials with unexpected functional properties. Oligopeptides can form amyloid-like cross-β assemblies that are able to emit intrinsic photoluminescence (PL), over the whole near-UV/visible range, whose origin is still largely debated. As proton transfer between the peptide chain termini within the assembly is one of the invoked interpretations of this phenomenon, we here evaluated the solid state PL properties of a series of self-assembled hexaphenylalanine peptides characterized by a different terminal charge state. Overall, our data indicate that the charge state of these peptides has a marginal role in the PL emission as all systems exhibit very similar multicolour PL associated with a violation of the Kasha’s rule. On the other hand, charged/uncharged ends occasionally produce differences in the quantum yields. The generality of these observations has been proven by extending these analyses to the Aβ16–21 peptide. Collectively, the present findings provide useful information for deciphering the code that links the spectroscopic properties of these assemblies to their structural/electronic features.

Acetylenic Bond Array Drives the Directional Assembly of Amyloid-Like Peptides on Graphyne

Acetylenic Bond Array Drives the Directional Assembly of Amyloid-Like Peptides on Graphyne

Fluorescence Emission of Self-assembling Amyloid-like Peptides: Solution versus Solid State.

Analysis of the intrinsic UV‐visible fluorescence exhibited by self‐assembling amyloid‐like peptides in solution and in solid the state highlights that their physical state has a profound impact on the optical properties. In the solid state, a linear dependence of the fluorescence emission peaks as a function of excitation wavelength is detected. On the contrary, an excitation‐independent emission is observed in solution. The present findings constitute a valuable benchmark for current and future explanations of the fluorescence emission by amyloids.

Thermo-Responsive self-assembly of a dual glucagon-like peptide and glucagon receptor agonist

The human peptide hormone Oxyntomodulin (Oxm) is known to induce satiety, increase energy expenditure, and control blood glucose in humans, making it a promising candidate for treatment of obesity and/or type 2 diabetes mellitus. However, a pharmaceutical exploitation has thus far been impeded by fast in vivo clearance and the molecule‘s sensitivity to half-life extending structural modifications. We recently showed that Oxm self-assembles into amyloid-like nanofibrils that continuously release active, soluble Oxm in a peptide-deprived environment. S.c. injected Oxm nanofibrils extended plasma exposure from a few hours to five days in rodents, compared to s.c. applied soluble Oxm. Here we show that Oxm fibril elongation kinetics and thermodynamics display a uniquely low temperature optimum compared to previously reported amyloid-like peptide and protein assemblies. Elongation rate is optimal at room temperature, with association rates 2–3 times higher at 25 °C than at ≥37 °C or ≤20 °C. We deduce from a combination of Cryo electron microscopy and spectroscopic methods that Oxm fibrils have a double-layered, triangular cross-section composed of arch-shaped monomers. We suggest a thermodynamic model that links the necessary molecular rearrangements during fibrillation and peptide release to the unique temperature effects in Oxm self-assembly and disassembly.

Binding Mechanisms of Amyloid-like Peptides to Lipid Bilayers and Effects of Divalent Cations.

In several neurodegenerative diseases, cell toxicity can emerge from damage produced by amyloid aggregates to lipid membranes. The details accounting for this damage are poorly understood including how individual amyloid peptides interact with phospholipid membranes before aggregation. Here, we use all-atom molecular dynamics simulations to investigate the molecular mechanisms accounting for amyloid-membrane interactions and the role played by calcium ions in this interaction. Model peptides known to self-assemble into amyloid fibrils and bilayer made from zwitterionic and anionic lipids are used in this study. We find that both electrostatic and hydrophobic interactions contribute to peptide-bilayer binding. In particular, the attraction of peptides to lipid bilayers is dominated by electrostatic interactions between positive residues and negative phosphate moieties of lipid head groups. This attraction is stronger for anionic bilayers than for zwitterionic ones. Hydrophobicity drives the burial of nonpolar residues into the interior of the bilayer producing strong binding in our simulations. Moreover, we observe that the attraction of peptides to the bilayer is significantly reduced in the presence of calcium ions. This is due to the binding of calcium ions to negative phosphate moieties of lipid head groups, which leaves phospholipid bilayers with a net positive charge. Strong binding of the peptide to the membrane occurs less frequently in the presence of calcium ions and involves the formation of a "Ca2+ bridge".

A Multidisciplinary Approach to Design Amyloid-Like Peptides to Form Supramolecular Assemblies

Specific sections of amyloid proteins, present in diseases such as Alzheimer's and Parkinson's, have recently inspired the design of peptides that self-assemble into supramolecular nanostructures. This work presents novel amyloid-like amphiphilic peptides that self-assemble into robust fibers with a beta-sheet like structure, in which the self-assembly is driven by π-stacking of aromatic residues placed at specific locations of the peptides. Different amyloid-like peptides have been designed in order to study how additional negatively charged residues at both ends of the sequence affect the morphology of the supramolecular structures.

Toward Understanding Optical Properties of Amyloids: A Reaction Path and Nonadiabatic Dynamics Study.

Amyloids have unique structural, chemical, and optical properties. Although much theoretical effort has been directed toward understanding amyloid nucleation, the understanding of their optical properties has remained rather limited. In particular, the photophysical mechanisms leading to near-UV excitation and characteristic blue-green luminescence in amyloid systems devoid of aromatic amino acids have not been resolved. We use ab initio static calculations and nonadiabatic dynamics simulations to study the excited electronic states of model amyloid-like peptides. We show that their photophysics is essentially governed by the multitude of nπ* states with excitation localized on the amide groups. The strong stabilization of the nπ* states with respect to the amide group deplanarization and the concomitant increase of the oscillator strength make excitation in the near-UV possible. With respect to emission, our dynamics simulations revealed that the amyloid cross β arrangement effectively hinders the nonradiative relaxation channels usually operative in proteins. Finally, we show that after relaxation of the photoexcited peptides toward the minimum of the different nπ* states, fluorescence takes place in the visible (green) part of the electromagnetic spectrum.

Role of the Metal Center in the Modulation of the Aggregation Process of Amyloid Model Systems by Square Planar Complexes Bearing 2-(2'-pyridyl)benzimidazole Ligands.

The effect of analogue Pd(II)-, Pt(II)-, and Au(III) compounds featuring 2-(2′-pyridyl)benzimidazole on the aggregation propensity of amyloid-like peptides derived from Aβ and from the C-terminal domain of nucleophosmin 1 was investigated. Kinetic profiles of aggregation were evaluated using thioflavin binding assays, whereas the interactions of the compounds with the peptides were studied by UV-Vis absorption spectroscopy and electrospray ionization mass spectrometry. The results indicate that the compounds modulate the aggregation of the investigated peptides using different mechanisms, suggesting that the reactivity of the metal center and the physicochemical properties of the metals (rather than those of the ligands and the geometry of the metal compounds) play a crucial role in determining the anti-aggregation properties.

Amyloid-like peptide nanofibrils as scaffolds for tissue engineering: Progress and challenges (Review)

Networks of amyloid-like nanofibrils assembled from short peptide sequences have the ability to form scaffolds that can encapsulate clinically relevant stem cells encouraging their attachment, growth, and differentiation into various lineages which can be used in tissue engineering applications to treat a range of diseases and traumas. In this review, the author highlights a selection of important proof-of-principle papers that show how this class of self-assembled networks is highly suited to biomaterial scaffold development. The author highlights recent studies which have shown that these scaffolds can be used to promote cell and tissue regeneration both in vitro and in vivo. The author also presents some fundamental knowledge gaps which are preventing the widespread translation of such scaffolds. Finally, the author outlines a selection of studies that elucidate molecular assembly mechanisms and biophysical properties of amyloid-like peptide nanofibrils and suggests how studies like these might lead to the ability to generate nanofibril scaffolds with bespoke properties for tissue engineering.

Self-assembling amyloid-like peptides as exogenous second harmonic probes for bioimaging applications.

Amyloid-like peptides are an ideal model for the mechanistic study of amyloidosis, which may lead to many human diseases, such as Alzheimer disease. This study reports a strong second harmonic generation (SHG) effect of amyloid-like peptides, having a signal equivalent to or even higher than those of endogenous collagen fibers. Several amyloid-like peptides (both synthetic and natural) were examined under SHG microscopy and shown they are SHG-active. These peptides can also be observed inside cells (in vitro). This interesting property can make these amyloid-like peptides second harmonic probes for bioimaging applications. Furthermore, SHG microscopy can provide a simple and label-free approach to detect amyloidosis. Lattice corneal dystrophy was chosen as a model disease of amyloidosis. Morphological difference between normal and diseased human corneal biopsy samples can be easily recognized, proving that SHG can be a useful tool for disease diagnosis.

Unraveling the Design Rules in Ultrashort Amyloid-Based Peptide Assemblies toward Shape-Controlled Synthesis of Gold Nanoparticles.

The fundamental understanding of the detailed relationship between molecular structure and material function remains a challenging task, until now. In order to understand the relative contribution of aromatic moieties and hydrophobicity of amino acid chains, we designed a library of ultrashort amyloid-like peptides based on Ar-Phe-X (where "Ar" represents different aromatic moieties and "X" represents amino acids having varied side-chain functionalities). Our research clearly indicated that the alteration in the size and hydrophobicity of the aromatic capping play a crucial role compared to the subtle change in the amino acid sequence of the dipeptide in dictating the final self-assembled structure and properties of these short peptide amphiphiles. Further, we explored our detailed understanding toward the controlled synthesis of bioinspired organic-inorganic hybrids. For the first time, we established the differential role of aliphatic and aromatic hydroxyl moieties toward the in situ shape-controlled synthesis of gold nanoparticles in three-dimensional nanostructures of hydrogels. To the best of our knowledge, it is the first report which demonstrated the formation of rectangular platonic gold nanoparticles using simple dipeptide hydrogels, exhibiting pH-dependent size control. Our study shows promising implications in bottom-up nanofabrication of next-generation nanomaterials with emergent properties.

Neglected Hydrophobicity of Dimethanediyl Group in Peptide Self-Assembly: A Hint from Amyloid-like Peptide GNNQQNY and Its Derivatives.

Besides typical hydrophobic amino acids providing hydrophobic interactions, glutamine as a hydrophilic amino acid has also been known to be an important element in many self-assembling peptides, but it is still not clear how this particular amino acid contributes to the self-assembling process. We supposed that the dimethanediyl group in the side chain of glutamine could provide hydrophobic interaction for peptide self-assembly. To prove this hypothesis, we used the GNNQQNY peptide and its derivatives as examples to show the importance of the dimethanediyl group for peptide self-assembly. We found a very close relationship between the number of dimethanediyl groups, the strength of hydrophobic interaction, and the self-assembling ability of the peptides, indicating the hydrophobicity of the dimethanediyl group and its important role for self-assembly. This new finding might be instructive for clarifying the self-assembling mechanism of many natural peptides, as well as for developing novel self-assembling peptide nanomaterials.

Generation and Characterization of Virus-Enhancing Peptide Nanofibrils Functionalized with Fluorescent Labels.

Retroviral gene transfer is the method of choice for the stable introduction of genetic material into the cellular genome. However, efficient gene transfer is often limited by low transduction rates of the viral vectors. We have recently described a 12-mer peptide, termed EF-C, that forms amyloid-like peptide nanofibrils (PNF), strongly increasing viral transduction efficiencies. These nanofibrils are polycationic and bind negatively charged membranes of virions and cells, thereby overcoming charge repulsions and resulting in increased rates of virion attachment and gene transfer. EF-C PNF enhance vector transduction more efficiently than other soluble additives and offer prospects for clinical applications. However, while the transduction-enhancing activity of PNF has been well-characterized, the exact mechanism and the kinetics underlying infection enhancement as well as the cellular fate of the fibrils are hardly explored. This is partially due to the fact that current labeling techniques for PNF rely on amyloid probes that cause high background staining or lose signal intensities after cellular uptake. Here, we sought to generate EF-C PNF covalently coupled with fluorescent labels. To achieve such covalent bioconjugates, the free amino groups of the EF-C peptide were coupled to the ATTO 495 or 647N NHS ester dyes. When small amounts of the labeled peptides were mixed with a 100- to 10 000-fold excess of the native peptide, PNF formed that were morphologically indistinguishable from those derived from the unlabeled peptide. The fluorescence of the fibrils could be readily detected using fluorescence spectroscopy, microscopy, and flow cytometry. In addition, labeled and nonlabeled fibrils captured viral particles and increased retroviral transduction with similar efficacy. These covalently fluorescence-labeled PNF are valuable tools with which to elucidate the mechanism(s) underlying transduction attachment and the fate of the fibrils in cells, tissues, and animal models.

Molecular Mechanism of Surface-Assisted Self-Assembly of Amyloid-Like Peptides

Peptide self-assembly attracted huge interests due to its clinical importance in protein-aggregation diseases, as well as potential applications for novel material design. Large efforts have been exerted on unraveling factors for the growth dynamics and morphologies. However, an intrinsic delicacy present in molecular systems and environmental elements, such as electrophysical properties of surface and solution, often results in controversy. Recently, a 9-residue long peptide designed from consensus sequences of amyloidogenic proteins was shown to assemble into a highly-ordered epitaxial structure on both hydrophilic mica and hydrophobic highly-oriented pyrolytic graphite (HOPG) surfaces but with very different morphologies: upright conformations on mica and lying-down on HOPG. Furthermore, with raised salt concentrations the peptides formed a highly-ordered multilayered nanofilaments at the mica/water interface in highly controllable fashion, which is unusual compared to uncontrollable, disordered, amorphous aggregates observed in other proteins in the similar condition. In our study, we investigated in molecular level details on how various environmental factors determine the peptide assemblies on mica and HOPG surfaces from their morphologies to the epitaxial growth mechanism, using atomistic molecular dynamics simulations. Firstly, not only surface polarity but also surface structure closely incorporate with individual peptide structure, as synergistically inducing a highly-optimized parallel β-stranded arrays on mica. Next, we showed that hydrophobic sidechain interaction indeed drives the longitudinal length growth of the assembly, while hydrophilic backbone hydrogen bonds rather control the transversal thickness. Finally, in raised salt concentration, we found that double-layered structures of all upright conformation are energetically favorable on mica but with anti-parallel β-stands for the upper layer, while the lower still in the parallel configuration, which again emphasizes the importance of environmental factors like contacting surface.

Self-Assembly of Amyloid-Like Peptides at Interfaces Investigated by Atomic Force Microscopy

In the past few years, numerous researches have focused on the self-assembly of amyloid peptides and proteins. Especially, the self-assembly of peptides and proteins at interfaces has attracted much attention, on account of the potential applications as well as understanding mechanism of some neurodegenerative diseases. In this review, we centered topic on recent progress of the self-assembly of amyloid-like peptides at interfaces, which was mainly carried out in our group by employing atomic force microscopy (AFM). In the first part of the review, we introduced how the amyloid peptides assembled at the solid substrate/water interface. And nanoscale control of the assembly of the peptides by AFM has also been introduced. In the second part of this review, we focused on how the amyloid-like peptides assembled at air/solid interface, in which diffusion and self-assembly of peptides occurred with the help of a water nanofilm confined from ambient environment onto the solid surface. These results indicate that interfaces play an important role in peptide assembly, and also prove that AFM is one of the powerful instruments in studying peptide self-assembly.