Formation Of Amyloid-like Fibrils Research Articles

Amyloidogenesis of SARS-CoV-2 delta plus and omicron variants receptor-binding domain (RBD): impact of SUMO fusion tag.

The RBD of SARS-CoV-2 mediates viral entry into host cells by binding to the host receptor ACE2. SARS-CoV-2 infection is linked to various health issues resembling amyloid-related problems, persuading us to investigate the amyloidogenicity of the SARS-CoV-2 spike RBD. The FoldAmyloid program was used to assess the amyloidogenic propensities in the RBD of Delta Plus and RBD of the Omicron variant, with and without the SUMO tag. After the expression of RBDs, purification, and dialysis steps were performed, subsequently the ThT assay, FTIR, and TEM were employed to check the RBD ability to form fibrils. The ThT assay, TEM, and FTIR revealed the ability of RBD to self-assemble into β-sheet-rich aggregates (48.4% β-sheet content). Additionally, the presence of the SUMO tag reduced the formation of RBD amyloid-like fibrils. The amyloidogenic potential of Omicron RBD was higher than Delta Plus, according to both in silico and experimental analyses. The SARS-CoV-2 RBD can assemble itself by forming aggregates containing amyloid-like fibrils and the presence of a SUMO tag can significantly decrease the formation of RBD amyloid-like fibrils. In silico analysis suggested that variation in the ThT fluorescence intensity of amyloid accumulations in the two SARS-CoV-2 strains arises from specific mutations in their RBD regions.

Atomic force microscopy of spherical intermediates on the pathway to fibril formation of influenza A virus nuclear export protein.

The nuclear export protein of the influenza A virus (NEP) is involved in many important processes of the virus life cycle. This makes it an attractive target for the treatment of a disease caused by a virus. Previously it has been shown, that recombinant variants of NEP are highly prone to aggregation in solution under various conditions with the formation of amyloid-like aggregates. In the present work, the amyloid nature of NEP aggregates was evidenced by Congo red binding assays. Atomic force microscopy has shown that NEP can form two types of spherical nanoparticles, which provide an alternative pathway for the formation of amyloid-like fibrils. Type I of these "fibrillogenic" spheres, formed under physiological conditions, represents the micelle-like particles with height 10-60 nm, which can generate worm-like flexible fibrils with the diameter 2.5-4.0 nm, length 20-500 nm and the Young's modulus ~73 MPa. Type II spherical aggregates with size of about 400-1000 nm, formed at elevated temperatures, includes fractions of drop-like and vesicle-like particles, generating more rigid amyloid-like fibrils with height of ~8 nm, and length of up to 2 μm. The hypothetical mechanism of fibril formation via nanospherical structures was suggested. RESEARCH HIGHLIGHTS: AFM has revealed two types of the influenza A virus nuclear export protein spherical aggregates. They provide an alternative pathway for the formation of amyloid-like fibrils. The mechanism of fibril formation via spherical structures is suggested.

Ultrasonic pretreatment and epigallocatechin gallate incorporation enhance the formation, apparent viscosity, and antioxidant activity of pea protein amyloid-like fibrils

Research on the application of plant protein-based carrier in the field of food is increasing in recent years. Pea protein, a critical plant protein source in food processing, has the advantages of high nutritional value, low price and low allergy. In this study, the effects of pre-ultrasonic treatment on the fibrillation behavior of pea protein, the microstructure, rheological property and antioxidant activity of pea protein amyloid-like fibrils as well as the interaction between epigallocatechin gallate and pea protein amyloid-like fibrils were examined. Thioflavin T fluorescence illuminated pre-ultrasonic treatment could dramatically facilitate the formation of β-sheet structure. Interaction between epigallocatechin gallate and pea protein fibrils was mainly through hydrophobic interaction, evidenced by intrinsic fluorescence spectroscopy, X-ray diffraction and surface hydrophobicity analysis. Antioxidant properties and apparent viscosity of pea protein fibrils were remarkably enhanced with both ultrasound and epigallocatechin gallate addition. The pre-ultrasound treated amyloid-like fibrils were more slender than those without treatment in terms of morphology. It was found that pre-ultrasound treated pea protein fibrils-epigallocatechin gallate composites exhibited the highest stability, viscosity and antioxidant activities, which showed great promising as excellent carriers of bioactive substances. The results obtained in this study could enable the utilization of pea protein fibrils-based hydrogel for stabilizing, encapsulating, and delivering bioactive compounds in food processing.

Multiscale simulations reveal TDP-43 molecular-level interactions driving condensation

The RNA-binding protein TDP-43 is associated with mRNA processing and transport from the nucleus to the cytoplasm. TDP-43 localizes in the nucleus as well as accumulating in cytoplasmic condensates such as stress granules. Aggregation and formation of amyloid-like fibrils of cytoplasmic TDP-43 are hallmarks of numerous neurodegenerative diseases, most strikingly present in >90% of amyotrophic lateral sclerosis (ALS) patients. If excessive accumulation of cytoplasmic TDP-43 causes, or is caused by, neurodegeneration is presently not known. In this work, we use molecular dynamics simulations at multiple resolutions to explore TDP-43 self- and cross-interaction dynamics. A full-length molecular model of TDP-43, all 414 amino acids, was constructed from select structures of the protein functional domains (N-terminal domain, and two RNA recognition motifs, RRM1 and RRM2) and modeling of disordered connecting loops and the low complexity glycine-rich C-terminus domain. All-atom CHARMM36m simulations of single TDP-43 proteins served as guides to construct a coarse-grained Martini 3 model of TDP-43. The Martini model and a coarser implicit solvent C⍺ model, optimized for disordered proteins, were subsequently used to probe TDP-43 interactions; self-interactions from single-chain full-length TDP-43 simulations, cross-interactions from simulations with two proteins and simulations with assemblies of dozens to hundreds of proteins. Our findings illustrate the utility of different modeling scales for accessing TDP-43 molecular-level interactions and suggest that TDP-43 has numerous interaction preferences or patterns, exhibiting an overall strong, but dynamic, association and driving the formation of biomolecular condensates.

Investigation of serum amyloid a within animal species focusing on the 1-25 amino acid region

AA amyloidosis, characterized by the misfolding of serum amyloid A (SAA) protein, is the most common amyloid protein disorder across multiple species. SAA is a positive-acute phase protein synthesized by the liver in response to inflammation or stress, and it normally associates with high-density lipoprotein at its N-terminus. In this study, we focused on the 1-25 amino acid (aa) region of the complete 104 aa SAA sequence to examine the aggregation propensity of AA amyloid. A library comprising eight peptides from different species was assembled for analysis. To access the aggregation propensity of each peptide region, a bioinformatic study was conducted using the algorithm TANGO. Congo red (CR) binding assays, Thioflavin T (ThT) assays, and transmission electron microscopy (TEM) were utilized to evaluate whether the synthesized peptides formed amyloid-like fibrils. All synthetic SAA 1-25 congeners resulted in amyloid-like fibrils formation (per CR and/or ThT staining and TEM detection) at the exception of the ferret SAA1-25 fragment, which generated plaque-like materials by TEM. Ten residues were preserved among SAA 1-25 congeners resulting in amyloid-like fibrils, i.e. F6, E9, A10, G13, D16, M17, A20, Y21, D23, and M24. Amino acid residues highlighted by this study may have a role in increasing the propensity for amyloid-like fibril formation. This study put an emphasis on region 1-25 in the mechanism of SAA1 misfolding.

Formation and structural characteristics of pea globulin amyloid-like fibrils pretreated with low-frequency magnetic field

In this study, the effect of low-frequency magnetic field (LF-MF) on the formation and structural characteristics of pea globulin (PG) amyloid-like fibrils were investigated. The results showed that the structure of PG unfolded after LF-MF pretreatment. And PG pretreated with LF-MF (MPG) exhibited higher thioflavin T fluorescence intensity during the fibrillation compared with native PG (NPG), indicating that LF-MF could enhance the ability of PG to form amyloid-like fibrils. During the fibrillation process, the PG was hydrolyzed into small peptides in the initial stage of heating, leading to decreased particle size, as evidenced by the degraded subunits. The average particle size then increased with the small peptides aggregating, and the ordered amyloid-like fibrils with high content of β-sheets were formed. But the fibrils generated by MPG were larger in size and had a higher content of β-sheets. The formation of PG amyloid-like fibrils was confirmed by AFM images, and LF-MF pretreatment resulted in the generation of longer and thicker PG fibrils. The results of surface hydrophobicity showed that the hydrophobic interactions played an important role in the fibrillation process of PG. This work may provide a deep understanding about the effect of LF-MF on the assembly behavior and structural characteristics of PG amyloid-like fibrils.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111–142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111–142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111–142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111–142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111–142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Biomineralization in Barnacle Base Plate in Association with Adhesive Cement Protein.

Barnacles strongly attach to various underwater substrates by depositing and curing a proteinaceous cement that forms a permanent adhesive layer. The protein MrCP20 present within the calcareous base plate of the acorn barnacle Megabalanus rosa (M.rosa) was investigated for its role in regulating biomineralization and growth of the barnacle base plate, as well as the influence of the mineral on the protein structure and corresponding functional role. Calcium carbonate (CaCO3) growth on gold surfaces modified by 11-mercaptoundecanoic acid (MUA/Au) with or without the protein was followed using quartz crystal microbalance with dissipation monitoring (QCM-D), and the grown crystal polymorph was identified by Raman spectroscopy. It is found that MrCP20 either in solution or on the surface affects the kinetics of nucleation and growth of crystals and stabilizes the metastable vaterite polymorph of CaCO3. A comparative study of mass uptake calculated by applying the Sauerbrey equation to the QCM-D data and quantitative X-ray photoelectron spectroscopy determined that the final surface density of the crystals as well as the crystallization kinetics are influenced by MrCP20. In addition, polarization modulation infrared reflection-absorption spectroscopy of MrCP20 established that, during crystal growth, the content of β-sheet structures in MrCP20 increases, in line with the formation of amyloid-like fibrils. The results provide insights into the molecular mechanisms by which MrCP20 regulates the biomineralization of the barnacle base plate, while favoring fibril formation, which is advantageous for other functional roles such as adhesion and cohesion.

OASL phase condensation induces amyloid-like fibrillation of RIPK3 to promote virus-induced necroptosis

RIPK3–ZBP1–MLKL-mediated necroptosis is a proinflammatory cell death process that is crucial for antiviral host defence. RIPK3 self-oligomerization and autophosphorylation are prerequisites for executing necroptosis, yet the underlying mechanism of virus-induced RIPK3 activation remains elusive. Interferon-inducible 2′-5′ oligoadenylate synthetase-like (OASL) protein is devoid of enzymatic function but displays potent antiviral activity. Here we describe a role of OASL as a virus-induced necroptosis promoter that scaffolds the RIPK3–ZBP1 non-canonical necrosome via liquid-like phase condensation. This liquid-like platform of OASL recruits RIPK3 and ZBP1 via protein–protein interactions to provide spatial segregation for RIPK3 nucleation. This process facilitates the amyloid-like fibril formation and activation of RIPK3 and thereby MLKL phosphorylation for necroptosis. Mice deficient in Oasl1 exhibit severely impaired necroptosis and attenuated inflammation after viral infection, resulting in uncontrolled viral dissemination and lethality. Our study demonstrates an interferon-induced innate response whereby OASL scaffolds RIPK3–ZBP1 assembly via its phase-separated liquid droplets to facilitate necroptosis-mediated antiviral immunity.

Inside and outside of virus-like particles HBc and HBc/4M2e: A comprehensive study of the structure

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.

Structural Requirement of hA5G18 Peptide (DDFVFYVGGYPS) from Laminin α5 Chain for Amyloid-like Fibril Formation and Cell Adhesion.

The hA5G18 peptide (DDFVFYVGGYPS) identified from the human laminin α5 chain G domain promotes cell attachment and spreading when directly coated on a plastic plate, but does not show activity when it is conjugated on a chitosan matrix. Here, we focused on the structural requirement of hA5G18 for activity. hA5G18 was stained with Congo red and formed amyloid-like fibrils. A deletion analysis of hA5G18 revealed that FVFYV was a minimum active sequence for the formation of amyloid-like fibrils, but FVFYV did not promote cell attachment. Next, we designed functional fibrils using FVFYV as a template for amyloid-like fibrils. When we conjugated an integrin binding sequence Arg-Gly-Asp (RGD) to the FVFYV peptide with Gly-Gly (GG) as a spacer, FVFYVGGRGD promoted cell attachment in a plate coat assay, but a negative control sequence RGE conjugated peptide, FVFYVGGRGE, also showed activity. However, when the peptides were conjugated to Sepharose beads, the FVFYVGGRGD beads showed cell attachment activity, but the FVFYVGGRGE beads did not. These results suggest that RGD and RGE similarly contribute to cell attachment activity in amyloid-like fibrils, but only RGD contributes the activity on the Sepharose beads. Further, we conjugated a basic amino acid (Arg, Lys, and His) to the FVFYV peptide. Arg or Lys-conjugated FVFYV peptides, FVFYVGGR and FVFYVGGK, showed cell attachment activity when they were coated on a plate, but a His-conjugated FVFYV peptide FVFYVGGH did not show activity. None of the basic amino acid-conjugated peptides showed cell attachment in a Sepharose bead assay. The cell attachment and spreading on FVFYVGGR and FVFYVGGK were inhibited by an anti-integrin β1 antibody. These results suggest that the Arg and Lys residues play critical roles in the interaction with integrins in amyloid-like fibrils. FVFYV is useful to use as a template for amyloid-like fibrils and to develop multi-functional biomaterials.

Impact of heat and enzymatic treatment on ovalbumin amyloid-like fibril formation and enzyme-induced gelation

Heating aqueous solutions of ovalbumin (OVA) may cause gel formation. When heated at pH conditions close to the protein's isoelectric point (towards neutral pH), turbid particulate gels are formed, whereas at acidic pH fine-stranded, transparent gels are formed already at lower concentrations. Here, transparent gels were formed when subjecting 2.0% OVA to a combined heat (78 °C for 22 h at pH 7) and trypsin (37 °C for 48 h) treatment. Transmission electron microscopy clearly revealed the presence of long, straight OVA fibrils which contributed to the gel formation. Quartz crystal microbalance with dissipation (adsorption of small structures), size exclusion – HPLC (presence of both structures larger and smaller than native OVA) and atomic force microscopy (presence of long fibrils with a higher thickness, whereas heated OVA mainly showed amorphous aggregates) analyses confirmed that the additional enzymatic treatment was able to break down the amorphous aggregates formed by heating OVA into peptides, which then partly re-assembled into longer OVA fibrils. The above mentioned heat and enzymatic treatment conditions brought about gelation after 17 h with a gel strength of 68 Pa which broke at a stress of 38 Pa. By varying the temperature during heat (58–88 °C) and enzymatic (27–67 °C) treatments, gels were formed the fastest when heated at 78 °C followed by enzymatic treatment at 57 °C. A design of experiments for evaluating the impact of OVA and trypsin concentration revealed that the fastest gelation occurred at the higher considered OVA and trypsin concentrations. Additionally, the gel strength was also higher under the latter conditions. It is clear that different gel characteristics can be reached when varying the different process conditions, creating the opportunity for reconsidering the formulation of various foods such as jellies, marmalades and desserts. • Combined heat and tryptic treatment of ovalbumin (OVA) induces gelation. • Trypsin hydrolyzes amorphous aggregates of OVA. • The resultant peptides can form amyloid-like fibrils. • Preheating and trypsin-incubation temperatures influence the gelation. • Increasing OVA and trypsin concentrations result in rapid formation of strong gels.

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform

Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.

Toxic SOD1 trimers are off-pathway in the formation of amyloid-like fibrils in ALS

Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. However, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super-stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron-like cell model (NSC-34). Using size exclusion chromatography, we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis.

Evaluation of the impact of stirring on the formation, structural changes and rheological properties of ovalbumin fibrils

The tendency to self-assemble into amyloid fibrils with an ordered cross-β structure is a generic feature of proteins. The formation mechanism of ovalbumin (OVA) fibrils at 85 °C and pH 2.0 under static or stirring condition was investigated in this study. The results showed that the formation of OVA amyloid-like fibrils was confirmed by Thioflavin T fluorescence, and the formation rate was accelerated by stirring treatment. The building blocks of OVA fibrils were peptides with molecular weight between 10 and 30 kDa, whether stirred or not. With the extension of heating time, more and more worm-like and flexible fibrils were observed by atomic force microscopy under stirring condition. The zeta potential results showed that OVA was positively charged in an acidic environment, which favored the formation of the ordered aggregates. Based on the results of circular dichroism, surface hydrophobicity and free –SH content, it could be concluded that both non-covalent interactions (hydrogen bonds and hydrophobic interactions) and covalent interactions (disulfide bonds) were involved in the formation of OVA fibrils. OVA fibrils formed under stirring condition showed a much higher viscosity and storage modulus than those formed under static condition. Furthermore, OVA fibrils showed no in vitro cytotoxicity, which has the potential for future applications. This work can further deepen the understanding of the formation mechanism of food protein amyloid-like fibrils.

Multiscale simulations reveal molecular dynamics of TDP-43 assemblies

The RNA binding protein TDP-43 is involved with mRNA processing and transport from the nucleus to the cytoplasm. TDP-43 localizes in the nucleus as well as accumulates in cytoplasmic intracellular condensates and stress granules. Aggregation and formation of amyloid-like fibrils of cytoplasmic TDP-43 are hallmarks of numerous neurodegenerative diseases, most strikingly present in >90% of amyotrophic lateral sclerosis (ALS) patients. How excessive accumulation of cytoplasmic TDP-43 causes, or is caused by, neurodegeneration is presently not known.

Colloidal stability of oil-in-water emulsions prepared from hen egg white submitted to dry and/or wet heating to induce amyloid-like fibril formation

Hen egg white (HEW) proteins are components of some foods and improvement of their techno-functional properties has been widely studied. During food processing, they are commonly subjected to heating. Here, both the impact of dry, wet, and combined dry and wet heat treatment was investigated. Heating (1 h at 80 °C) dried HEW (still containing 12% water) resulted in formation of glycated Maillard compounds such as protein-glucose conjugates, but only in limited amyloid-like fibril (ALF) formation. Wet heating (0.5 h at 80 °C, in excess water) clearly resulted in increased relative Thioflavin T (ThT) fluorescence, indicating the formation of ALFs. When dry heating (0.5 h at 80 °C) was followed by wet heating (24 h at 80 °C), the slightly lower relative ThT fluorescence than obtained when wet heating HEW suggested that ALF formation was limited due to protein glycation. When control (i.e. unheated) HEW dispersions and dispersions containing different heated HEW samples were used as continuous phase for oil-in-water emulsions, control HEW resulted in a multimodal particle size distribution and high creaming rate. In contrast, emulsions stabilized by dry and wet heated HEW showed an overall reduced volume-weighted average droplet diameter (D43) and increased creaming stability. HEW subjected to 4 h dry heat treatment at 80 °C and 24 h wet heat treatment at 80 °C resulted in emulsions with a monomodal particle size distribution with the smallest D43 (0.47 ± 0.01 μm), as well as the lowest creaming rate (68.4 ± 9.77 mm/day). Comparison of the relative ThT fluorescence, D43 and creaming rate by principal component analysis revealed that mainly the emulsions with dispersions having high relative ThT fluorescence had lower average particle sizes and consequently a higher creaming stability. Thus, formation of ALFs by wet heating of HEW seems to be the better heat treatment for improving their techno-functional properties.

Amphiphilic Histidine-Based Oligopeptides Exhibit pH-Reversible Fibril Formation.

We report the design, simulation, synthesis, and reversible self-assembly of nanofibrils using polyhistidine-based oligopeptides. The inclusion of aromatic amino acids in the histidine block produces distinct antiparallel β-strands that lead to the formation of amyloid-like fibrils. The structures undergo self-assembly in response to a change in pH. This creates the potential to produce well-defined fibrils for biotechnological and biomedical applications that are pH-responsive in a physiologically relevant range.

Isomerization of Asp is essential for assembly of amyloid-like fibrils of αA-crystallin-derived peptide.

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66–80 peptide, corresponding to the 66–80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66–80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66–80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact β-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.

Understanding the Formation of Apoferritin Amyloid Fibrils.

We present the optimization of experimental conditions to yield long, rigid apoferritin protein amyloid fibrils, as well as the corresponding fibrillation pathway. Fibril growth kinetics was followed using atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS), circular dichroism (CD), fourier-transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among the morphologies identified, we show that the conditions result in small aggregates, as well as medium and long fibrils. Extended incubation times led to progressive unfolding and hydrolysis of the proteins into very short peptide fragments. AFM, SDS-PAGE, and CD support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role. These collective results provide convincing evidence that protein unfolding and complete hydrolysis of the proteins into very short peptide sequences are essential for the formation of the final apoferritin amyloid-like fibrils.