Amyloid-like Fibers Research Articles

Utilization of peanut protein isolate amyloid-like fibers as stabilizers for high internal phase Pickering emulsions

This paper focuses on modifying peanut protein isolate (PPI) through fibrillation to enhance its emulsification properties and investigates the potential application of the fibers in stabilizing high internal phase Pickering emulsions (HIPPEs). Th T result indicates that the optimal conditions for peanut protein isolate amyloid-like fibers (PAFs) preparation are pH=2, 90 °C, and 20 h. Electrophoresis and Transmission electron microscope demonstrate that PPI undergoes peptide hydrolysis and self-assembly during the reaction process, leading to the formation of an elongated linear structure with a diameter approximately 14 nm. Furthermore, infrared spectroscopy, X-ray diffraction, and contact angle results further demonstrate an increase in β-sheet content, crystallinity, and surface hydrophilicity compared to PPI. Notably, the emulsification activity index (EAI) of PAFs (78.62 ± 1.68 m2/g) was found to be increased up to threefold compared to that of PPI (27.89 ± 1.98 m2/g). According to visual observation and microstructural analysis, the addition of 1% PAFs has been found to effectively stabilize HIPPEs (φdisp = 0.75) due to the presence of dense protective layers formed by PAFs. Additionally, the particle size results (D[3,2] = 17.02 ± 2.02 μm) also indicate that the oil droplets are tightly enveloped by the dense protective layers formed by PAFs. Moreover, rheological data indicates that HIPPEs stabilized using PAFs exhibit excellent solid-like viscoelastic behavior. Overall, these findings suggest that the utilization of PAFs has significant potential in enhancing the stability of HIPPEs.

Poly(d-amino acid) Nanoparticles Target Staphylococcal Growth and Biofilm Disassembly by Interfering with Peptidoglycan Synthesis.

d-Amino acids are signals for biofilm disassembly. However, unexpected metabolic pathways severely attenuate the utilization of d-amino acids in biofilm disassembly, resulting in unsatisfactory efficiency. Herein, three-dimensional poly(d-amino acid) nanoparticles (NPs), which possess the ability to block intracellular metabolism, are constructed with the aim of disassembling the biofilms. The obtained poly(α-N-acryloyl-d-phenylalanine)-block-poly(β-N-acryloyl-d-aminoalanine NPs (denoted as FA NPs) present α-amino groups and α-carboxyl groups of d-aminoalanine on their surface, which guarantees that FA NPs can effectively insert into bacterial peptidoglycan (PG) via the mediation of PG binding protein 4 (PBP4). Subsequently, the FA NPs trigger the detachment of amyloid-like fibers that connect to the PG and reduce the number of polysaccharides and proteins in extracellular polymeric substances (EPS). Finally, FA NPs damage the structural stability of EPS and lead to the disassembly of the biofilm. Based on this feature, FA NPs significantly enhance the killing efficacy of encapsulated sitafloxacin sesquihydrate (Sita) by facilitating the penetration of Sita within the biofilm, achieving complete elimination of Staphylococcal biofilm in mice. Therefore, this study strongly demonstrates that FA NPs can effectively improve biofilm disassembly efficacy and provide great potential for bacterial biofilm infection treatment.

Lipid-induced polymorphic amyloid fibril formation by α-synuclein.

Many proteins that self-assemble into amyloid and amyloid-like fibers can adopt diverse polymorphic forms. These forms have been observed both in vitro and in vivo and can arise through variations in the steric-zipper interactions between β-sheets, variations in the arrangements between protofilaments, and differences in the number of protofilaments that make up a given fiber class. Different polymorphs arising from the same precursor molecule not only exhibit different levels of toxicity, but importantly can contribute to different disease conditions. However, the factors which contribute to formation of polymorphic forms of amyloid fibrils are not known. In this work, we show that in the presence of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, a highly abundant lipid in the plasma membrane of neurons, the aggregation of α-synuclein is markedly accelerated and yields a diversity of polymorphic forms under identical experimental conditions. This morphological diversity includes thin and curly fibrils, helical ribbons, twisted ribbons, nanotubes, and flat sheets. Furthermore, the amyloid fibrils formed incorporate lipids into their structures, which corroborates the previous report of the presence of α-synuclein fibrils with high lipid content in Lewy bodies. Thus, the present study demonstrates that an interface, such as that provided by a lipid membrane, can not only modulate the kinetics of α-synuclein amyloid aggregation but also plays an important role in the formation of morphological variants by incorporating lipid molecules in the process of amyloid fibril formation.

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain.

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

The Importance of MicroRNA Expression in Pseudoexfoliation Syndrome.

Pseudoexfoliation syndrome (PEX) is an important systemic disorder of the extracellular matrix, in which granular amyloid-like protein fibers accumulate in the anterior segment of the eyeball as well as in other organs. PEX is currently considered to be a multifactorial systemic disorder with genetic and environmental risk factors. The aim of this manuscript was to analyze miR expression in PEX. In recent years, an attempt has been made to investigate and describe the level of expression of selected miRs in PEX. Four polymorphisms of genes isolated from the blood that may be related to PEX were identified and miR-122-5p was found to be upregulated in patient blood. Furthermore, 18 miRs were identified with a statistically different expression in the aqueous humor. A significantly elevated expression of miR-125b was found in the anterior lens capsule, and four miRs were described, which may have a significant impact on the development of PEX. Regulatory miR molecules are gaining more and more importance in research aimed at identifying and isolating molecular markers related to the pathogenesis and prognosis of PEX, but further studies are needed.

Liquid-cell transmission electron microscopy for imaging of thermosensitive recombinant polymers

Various polymers used in controlled release applications exhibit solution-based thermal responses. Unfortunately, very few characterization and imaging techniques permit resolution of individual polymers during their thermally-triggered phase transitions. Here, we demonstrate the use of temperature-ramp liquid-cell transmission electron microscopy (LCTEM) for real-time evaluation of the solution and interfacial behavior of elastinlike polypeptides (ELPs) and their self-assembled nanostructures over a temperature range incorporating their intrinsic lower critical solution temperatures (LCSTs). Individual polymers and supramolecular assemblies were discriminated dependent upon solubility states. The recombinant polymers were shown to adsorb to the silicon-nitride chip window from the buffered saline solution and desorb in a temperature-dependent manner. Silk-elastinlike protein block copolymers (SELPs) (composed of repeat peptide motifs of silk and elastin) differed from ELPs in thermal behavior. While both polymers were shown to cluster, only SELPs formed robust amyloid-like fibers upon heating.

Modulating defibrillation by tryptophan-mediated photo cleavage of disulfide bonds

Dibenzyloxycarbonyl-l-cystine forms amyloid-like fibers and a hydrogel; however, on UV irradiation, Trp-mediated disulfide bond cleavage occurs, leading to defibrillation and a gel to sol transition.

Антибиопленочная активность антиматриксных молекул

Внеклеточный матрикс биопленок обеспечивает фиксацию биопленки на биологической поверхности и защиту ее бактерий от внешних неблагоприятных факторов. Основным структурным компонентом био­пленок является внеклеточное полисахаридное вещество. Ключевыми молекулярными структурами, поддерживающими трехмерную структуру биопленки, считают экзополисахариды и амилоидоподобные волокна. До недавнего времени предполагалось, что большинство механизмов диспергирования биопленок ассоциировано с функционированием матриксных деградирующих ферментов, таких как гликозидгидролазы, полисахаридные лиазы и протеазы. Однако продемонстрировано, что в процессе разрушения матриксных экзополисахаридов и амилоидоподобных волокон играют самостоятельную роль малые молекулы. Среди соединений, нарушающих матрикс биопленки, различают антиматриксные молекулы, соединения, взаимодействующие с микродоменами бактериальной мембраны, и бактериальные поверхностно-активные вещества (биосурфагенты). Продемонстрировано, что соединения данных групп могут ингибировать образование биопленок и способствовать их диспергированию. Из группы антиматриксных молекул полиаминное соединение норспермидин взаимодействует с экзополисахаридами, а производные бензохинона AA-861 и сесквитерпеновый лактон партенолид — с TasA-амилоидоподобными волокнами. Норспермидин предотвращает образование и диспергирует биопленки различных бактерий, включая Acinetobacter baumannii, Bacillus subtili, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis. Соединение AA-861 проявляет активность против биопленок, которые образованы бактериями Streptococcus mutans, Bacillus cereus, Escherichia coli, а партенолид диспергирует биопленки, сформированные Escherichia coli и Bacillus cereus. Сарагосовые кислоты, взаимодействуя с микродоменами бактериальной мембраны, нарушают функционирование рафт-ассоциированных протеинов бактерий. Малые антиматриксные молекулы и нацеленные на микродомены мембраны бактерий, инициирующие диспергирование биопленки, безусловно, станут основанием для разработки эффективных антибиопленочных терапевтических лекарственных средств.

Functionalized Prion-Inspired Amyloids for Biosensor Applications.

Protein amyloid nanofibers provide a biocompatible platform for the development of functional nanomaterials. However, the functionalities generated up to date are still limited. Typical building blocks correspond to aggregation-prone proteins and peptides, which must be modified by complex and expensive reactions post-assembly. There is high interest in researching alternative strategies to tailor amyloid-based nanostructures’ functionality on demand. In the present study, the biotin-streptavidin system was exploited for this purpose. Prion-inspired heptapeptides (Ac-NYNYNYN-NH2, Ac-QYQYQYQ-NH2, and Ac-SYSYSYS-NH2) were doped with biotin-conjugated counterparts and assembled into amyloid-like fibers under mild conditions. The scaffolds’ versatile functionalization was demonstrated by decorating them with different streptavidin conjugates, including gold nanoparticles, quantum dots, and enzymes. In particular, they were functionalized with peroxidase or phosphatase activities using streptavidin conjugated with horseradish peroxidase and alkaline phosphatase, respectively. Modification of amyloid-like nanostructures has generally been restricted to the addition of a single protein moiety. We functionalized the fibrils simultaneously with glucose oxidase and horseradish peroxidase, coupling these activities to build up a nanostructured glucose biosensor. Overall, we present a simple, modular, and multivalent approach for developing amyloid-based nanomaterials functionalized with any desired combination of chemical and biological moieties.

PB1-F2 amyloid-like fibers correlate with proinflammatory signaling and respiratory distress in influenza-infected mice

PB1-F2 is a virulence factor of influenza A virus known to increase viral pathogenicity in mammalian hosts. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers. However, the correlation between PB1-F2 structures and the resulting inflammatory response is unknown. Here, we used synchrotron-coupled Fourier transform-IR and deep UV microscopies to determine the presence of PB1-F2 fibers in influenza A virus–infected mice. In order to study the correlation between PB1-F2 structure and the inflammatory response, transgenic mice expressing luciferase under the control of an NF-κB promotor, allowing in vivo monitoring of inflammation, were intranasally instilled with monomeric, fibrillated, or truncated forms of recombinant PB1-F2. Our intravital NF-κB imaging, supported by cytokine quantification, clearly shows the proinflammatory effect of PB1-F2 fibers compared with N-terminal region of PB1-F2 unable to fibrillate. It is noteworthy that instillation of monomeric PB1-F2 of H5N1 virus induced a stronger inflammatory response when compared with prefibrillated PB1-F2 of H1N1 virus, suggesting mechanisms of virulence depending on PB1-F2 sequence. Finally, using whole-body plethysmography to measure volume changes in the lungs, we quantified the effects of the different forms of PB1-F2 on respiratory parameters. Thus, we conclude that PB1-F2–induced inflammation and respiratory distress are tightly correlated with sequence polymorphism and oligomerization status of the protein.

Acid soluble extracellular matrix confers structural stability to marine Bacillus haynesii pellicle biofilms

In natural and engineered settings, bacteria predominantly thrive in biofilms, which are complex microbial communities embedded in a self-produced extracellular polymeric substances (EPSs) matrix. Pellicles are complex macroscopic biofilms floating at air-water interface. Though pellicle formation has been studied in detail in Bacillus subtilis, a soil bacterium, it is not reported in aquatic bacteria, which may use pellicle-growth as survival-strategy. This study shows that Bacillus haynesii isolated from a marine environment forms robust pellicle biofilms at air-water interface. B. haynesii pellicles showed complex architecture, involving dense cell-aggregates with interconnecting thread-like structures in an extracellular matrix. In situ staining by Alcian blue, Concanavalin A and ThioflavinT (ThT), respectively, localized acidic polymers, glycoconjugates and amyloid-like fibers in the pellicle. The pellicle was rigid and not disrupted by common EPS extraction protocols. Hence, a set of reagents and conditions were evaluated for solubilizing the EPS and pellicle. Acetic acid was able to effectively solubilize the structural EPS and pellicle structure. Acid soluble structural EPS contained chemical signatures for both proteins and carbohydrates, as revealed by elemental analysis, Fourier Transform Infrared Spectroscopy and Raman Spectroscopy. Ex situ staining of acid soluble EPS by ThT showed recovery of amyloid-forming proteins from pellicle. Results show that structural stability of the pellicle is mainly conferred by amyloid-like fibers of the EPS matrix. The robust pellicle-growth reported here may represent a survival-strategy in the aquatic bacterium. The findings reported here can support future research on biofilm structure, EPS matrix and its formation, which are critical for understanding how microbes thrive in natural and engineered settings.

The biofilm-associated surface protein Esp of Enterococcus faecalis forms amyloid-like fibers

Functional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced by Enterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed.

Long‐Range Fluorescence Propagation in Amyloidogenic β‐Sheet Films and Fibers

AbstractNewly developed PEGylated phenylalanine peptide derivatives (PEG‐F6) can self‐assemble to form hybrid multifunctional nanostructures of homogeneous thin polymer films and amyloid‐like fibers. Both adopt a β‐sheet architecture and acquire the unique properties of visible fluorescence (FL) found in β‐sheet amyloid fibrils also associated with neurodegenerative diseases. A new paradigm observed in PEG‐F6 amyloid‐like fibers is reported, which demonstrates unique FL and optical absorption (OA) covering the entire visible region. Despite the full overlap of the FL and OA spectra, the FL propagates for an unexpectedly large distance of hundreds of micrometers in these fibers. A new non‐waveguiding mechanism of FL light energy transfer in amyloidogenic fibers is proposed based on photon reabsorption theory. The discovered quasi‐white FL of PEG‐F6 β‐sheet amyloidogenic fibers and films consists of the newly observed green FL band along the known blue FL band. The proof‐of‐concept of long sub‐millimeter range FL propagation in specially grown amyloidogenic PEG‐F6 fiber‐photonic probes is reported. The results indicate that FL can propagate in nanoscale fibers and suggest biomedical applications for FL monitoring of disease‐related ultrathin amyloid fibrils and in integrated photonics for optogenetics.

The Upper Hand of the Otu Amyloid Fibers: Increasing Enzymatic Activity and Prolonging Lifespan.

The formation of amyloid fibers is usually associated with aging and neurodegeneration. In this issue of Molecular Cell, Ji etal. (2019) demonstrate that the deubiquitinase Otu coalesces into amyloid-like fibers to enhance its activity and ensure its optimum biological function.

Effect of Subtilisin-like Proteinase of Bacillus pumilus 3–19 on Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa is a common device-associated pathogen and also a model object in biofilm research. In this study biofilm growth dynamics of Pseudomonas aeruginosa RSC-2 strain on different culture media was described. It was shown that Mueller-Hinton broth is more suitable for biofilm growth than Lysogeny broth. In vitro biofilm surface was investigated using scanning electron microscopy. Staining with Congo red dye demonstrated that the observed strain can produce amyloid-like fibers that play important role in biofilms and are proposed to be a target for proteinase treatment. The effect of subtilisin-like proteinase of Bacillus pumilus 3–19, trypsin, and proteinase K on biofilm was studied. All of these proteinases can destroy biofilms depending on the time of incubation and enzyme activity. After 4 h of incubation, 18–47% of biofilms were destroyed, which can be linked to the resistance of amyloid proteins to proteolytic degradation. However, an increase in the incubation time (to 24 h) led to complete destruction of biofilm.

Hydrogel Formation by an Aromatic Analogue of a β-Amyloid Fragment, Aβ16–22: A Scaffold for 3D Cell Culture

A short 7-residue stretch from β-amyloid, Aβ16–22 (Ac-KLVFFAE-am), yields typical amyloid-like fibrils at neutral pH. The Phe–Phe cassette present in the peptide is believed to be critical for its self-assembly. We report that the aromatic analogue Aβ16–22(F20Y) forms self-supporting soft gels at concentrations ≥2 mM even though the end-capped parent peptide does not form hydrogel up to 20 mM (1.8% w/w) concentration. The hydrogel is made up of distinct amyloid-like fibers. The storage modulus of 20 mM gel is ∼3–5-fold higher than the loss modulus in the 2–3000 rad/s angular frequency range, indicating distinct elastic properties. The hydrogel supports the growth of rat pancreatic cells (RIN-5F), human embryonic kidney cells (HEK-293), baby hamster kidney cells (BHK-21), and human neuroblastoma cells (IMR-32). The cells grow in clusters as is anticipated in a three-dimensional matrix. The rat pancreatic cells produced insulin, suggesting that they are functional inside the gel.

Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum: implications for prolamin evolution.

In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD γ-zein locates at the interface between a core of alcohol-soluble α-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD γ-zein. 16-kD γ-zein originates from 27-kD γ-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD γ-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD γ-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two γ-zeins indicates that the N-terminal region of 16-kD γ-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs.

Cell wall associated protein TasA provides an initial binding component to extracellular polysaccharides in dual-species biofilm

Many bacteria in biofilm surround themselves by an extracellular matrix composed mainly of extracellular polysaccharide (EP), proteins such as amyloid-like fibers (ALF) and nucleic acids. While the importance of EP in attachment and acceleration of biofilm by a number of different bacterial species is well established, the contribution of ALF to attachment in multispecies biofilm remains unknown. The study presented here aimed to investigate the role of TasA, a precursor for ALF, in cell-cell interactions in dual-species biofilms of Bacillus subtilis and Streptococcus mutans. Expression of major B. subtilis matrix operons was significantly up-regulated in the presence of S. mutans during different stages of biofilm formation, suggesting that the two species interacted and modulated gene expression in each other. Wild-type B. subtilis expressing TasA adhered strongly to S. mutans biofilm, while a TasA-deficient mutant was less adhesive and consequently less abundant in the dual-species biofilm. Dextran, a biofilm polysaccharide, induced aggregation of B. subtilis and stimulated adhesion to S. mutans biofilms. This effect was only observed in the wild-type strain, suggesting that interactions between TasA and dextran-associated EP plays an important role in inter-species interactions during initial stages of multispecies biofilm development.

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers.

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.