Fibrillation Pathway Research Articles

The Implementation and Iteration of an Emergency Department Atrial Fibrillation Pathway to Improve Patient Care

The Implementation and Iteration of an Emergency Department Atrial Fibrillation Pathway to Improve Patient Care

Fibrillation of Human Serum Albumin Differentially Affected by Asp-, Arg-, and Tyr-Capped Gold Nanoparticles.

Fibrillation of proteins is associated with a number of debilitating diseases, including various neurodegenerative disorders. Prevention of the protein fibrillation process is therefore of immense importance. We investigated the effect of amino acid-capped AuNPs on the prevention of the fibrillation process of human serum albumin (HSA), a model protein. Amino acid-capped AuNPs of varying sizes and agglomeration extents were synthesized under physiological conditions. The AuNPs were characterized by their characteristic surface plasmon resonance (SPR), and their interactions with HSA were investigated through emission spectroscopy in addition to circular dichroism (CD) spectral analyses. Fluorescence lifetime imaging (FLIM) as well as transmission electron microscopy (TEM) were used to observe the fibrillar network. Thermodynamic and kinetic analyses from CD and fluorescence emission spectra provided insights into the fibrillation pathway adopted by HSA in the presence of capped AuNPs. Kinetics of the fibrillation pathway followed by ThT fluorescence emission confirmed the sigmoidal nature of the process. The highest cooperativity was observed in the case of Asp-AuNPs with HSA. This was in accordance with the ΔG value obtained from the CD spectral analyses, where Arg-AuNPs with HSA showed the highest positive ΔG value and Asp-AuNPs with HSA showed the most negative ΔG value. The study provides information about the potential use of conjugate AuNPs to monitor the fibrillation process in proteins.

Aromatic vs alicyclic: Hydrophobicity of the ionic liquid on protein stability and fibril formation

Ionic liquids (ILs) are environment-friendly solvents with potential applications in biochemical reactions and as pharmaceutical ingredients. They are used to stabilize proteins and act as anti-aggregation agents in which the hydrophobic effect of ILs is suggested to have a primary role. To understand the differences in the effect of aromatic and alicyclic cations of ILs on thermal stability and fibrillation propensity of proteins, the effect of two alicyclic (butyl-1-methylpyrrolidinium and butyl-1-methylpiperidinium) and two aromatic (butyl-4-methylpyridinium and butyl-3-methylimidazolium) ILs on lysozyme was investigated by spectroscopy, densitometry, and molecular dynamics simulation. The results suggest that all the ILs switch the fibrillation pathway from nucleation-independent to nucleation-dependent mechanism at ≥200 mM. The fibrillation is slowed down by 28–35-fold in 500 mM of ILs, exceptionally pyrrolidinium-IL delays the fibrillation by ∼70-fold. All the ILs destabilize the protein during thermal denaturation. Though in all cases the unfolding is enthalpy-driven, pyridinium-IL at higher concentrations exhibits an entropy-driven unfolding effect. The volumetric analysis shows a higher thermal expansion coefficient for Lyz in pyridinium-IL which might be due to the increased internal cavity volume of the protein upon binding of the IL. These results corroborate with the computational studies that the preferential interaction coefficient of aromatic-ILs is more, particularly at a higher concentration (200 mM). Four preferable binding sites for ILs on the protein could be identified that includes the amyloidogenic regions, β-hairpin and the hydrophobic cluster 5. Also, the ILs are found to preferably interact with the protein through their alicyclic/aromatic ring and alkyl sidechain. These results suggest that the binding of ILs to the amyloidogenic region inhibits the fibril formation which has a similar effect for both alicyclic and aromatic ILs whereas aromatic-ILs show a larger destabilization effect than alicyclic-ILs at higher concentrations.

On-Pathway Oligomer of Human Islet Amyloid Polypeptide Induced and Stabilized by Mechanical Rotation during Magic Angle Spinning Nuclear Magnetic Resonance.

Intermediates along the fibrillation pathway are generally considered to be the toxic species responsible for the pathologies of amyloid diseases. However, structural studies of these species have been hampered by heterogeneity and poor stability under standard aqueous conditions. Here, we report a novel methodology for producing stable, on-pathway oligomers of the human type-2 diabetes-associated islet amyloid polypeptide (hIAPP or amylin) using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant β-sheet content and the capability of seeding hIAPP fibrillation. We used MAS nuclear magnetic resonance to demonstrate that the nature of the species was sensitive to sample conditions, including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems.

Inherent Adaptivity of Alzheimer Peptides to Crowded Environments.

Amyloid β (Aβ) is the major constituent in senile plaques of Alzheimer's disease in which peptides initially undergo structural conversions to form elongated fibrils. The impact of crowding on the fibrillation pathways of Aβ40 and Aβ42 , the most common peptide isoforms are studied. PEG and Ficoll are used as model crowders to mimic a macromolecular enriched surrounding. The fibrillar growth is monitored with the help of ThT-fluorescence assays in order to extract two rates describing primary and secondary processes of nucleation and growth. Techniques as fluorescence correlation spectroscopy and analytical ultracentrifugation are used to discuss oligomeric states; fibril morphologies are investigated using negative-staining transmission electron microscopy. While excluded volume effects imposed by macromolecular crowding are expected to always increase rates of intermolecular interactions and structural conversion, a vast variety of effects are found depending on the peptide, the crowder, or ionic strength of the solution. While investigations of the obtained rates with respect to a reactant-occluded model are capable to display specific surface interactions with the crowder, the employment of crystallization-like models revealthe crowder-induced entropic gain with J mol-1 per volume fraction of thecrowder.

Implementation of an integrated emergency department acute atrial fibrillation pathway safely reduces cardioversions and hospitalisations: A comparative pre-post study.

Atrial fibrillation/flutter (AF/AFL) accounts for high rates of ED presentations and hospital admissions. There is increasing evidence to suggest that delaying cardioversion for acute uncomplicated AF is safe, and that many patients will spontaneously revert to sinus rhythm (SR). We conducted a before-and-after evaluation of AF/AFL management after a change in ED pathway using a conservative 'rate-and-wait' approach, incorporating next working day outpatient clinic follow-up and delayed cardioversion if required. We performed a before-and-after retrospective cohort study examining outcomes for patients who presented to the ED in Christchurch, New Zealand, with acute uncomplicated AF/AFL in the 1-year period before and after the implementation of a new conservative management pathway. A total of 360 patients were included in the study (182 'Pre-pathway' vs 178 'Post-Pathway'). Compared to the pre-pathway cohort, those managed under the new pathway had an 81.2% reduction in ED cardioversions (n = 32 vs n = 6), and 50.7% reduction in all cardioversions (n = 65 vs n = 32). There was a 31.6% reduction in admissions from ED (n = 54 vs n = 79). ED length of stay (3.9 h vs 3.8 h, net difference -0.1 h, 95% confidence interval [CI] -0.6 to 0.3), 1-year ED AF representation (32.4% vs 26.4%, net difference -6.0% [95% CI -16.0% to 3.9%]), 1-year ED ischaemic stroke presentation (2.2% in both groups) and 7-day all-cause mortality rates (hazard ratio 1.05 [95% CI 0.6 to 1.9]) were all similar. Using a conservative 'rate-and-wait' strategy with early follow-up for patients presenting to ED with AF/AFL can safely reduce unnecessary cardioversions and avoidable hospitalisations.

The Anthocyanidin Peonidin Interferes with an Early Step in the Fibrillation Pathway of α-Synuclein and Modulates It toward Amorphous Aggregates.

Parkinson's disease (PD) is characterized by progressive degeneration of the dopaminergic neurons in the brain, accompanied by the accumulation of proteinaceous inclusions, Lewy bodies (LB), mainly comprised of alpha synuclein (α-syn) aggregates. The heterogeneity and the transient nature of the intermediate species formed in the α-syn fibrillation pathway have made it difficult to develop an effective therapeutic intervention. Therefore, any therapeutic molecule that could prevent as well as treat PD would be of great interest. Anthocyanidins are natural flavonoid compounds that have been shown to have neuroprotective properties and to modulate factors that cause neuronal death. Herein, we have explored the modulation and inhibition of α-syn fibrillation by the anthocyanidins cyanidin, delphinidin, and peonidin using a number of biophysical and structural tools. α-Syn fibrillation monitored using thioflavin T (ThT) fluorescence and light scattering suggested concentration dependent inhibition of α-syn fibrillation by all the three anthocyanidins. While cyanidin and delphinidin induced the formation of oligomers and small fibrillar structures of α-syn, respectively, peonidin led to the formation of amorphous aggregates, as observed by Atomic Force Microscopy (AFM). Peonidin proved to be most effective of the three anthocyanidins toward alleviating cell toxicity of SH-SY5Y neuroblastoma cells at concentrations where α-synuclein fibrillation was completely suppressed. Hence, the inhibition mechanism of peonidin was further explored by studying its interaction with α-syn using titration calorimetry and molecular docking. The results show its weak binding (in mM range) to the NAC region of α-syn through hydrogen bonding interactions. Also, circular dichroism and Raman spectroscopy revealed the structural aspects of peonidin-induced α-syn amorphous aggregates showing alpha helical structures with exposed Phe and Tyr regions. Due to the neuroprotective nature of peonidin, the findings reported here are significant and can be further explored toward developing a modifying therapy that could address both disease onset as well as the progression of PD.

Factors predictive of spontaneous reversion to sinus rhythm: Findings from an integrated acute atrial fibrillation pathway

Recent evidence suggests that delayed cardioversion of uncomplicated acute AF is non-inferior to early cardioversion in the Emergency Department (ED), 1 Pluymaekers N.A.H.A. Dudink E.A.M.P. Luermans J.G.L.M. et al. Early or Delayed Cardioversion in Recent-Onset Atrial Fibrillation. N Engl J Med. 2019; 380: 1499-1508 Crossref PubMed Scopus (95) Google Scholar and most patients will revert spontaneously to sinus rhythm (SR) by a rate control strategy. 2 Abadie B.Q. Hansen B. Walker J. et al. Likelihood of Spontaneous Cardioversion of Atrial Fibrillation Using a Conservative Management Strategy Among Patients Presenting to the Emergency Department. Am J Cardiol. 2019; 124: 1534-1539 Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar We adopted a new inter-departmental acute AF clinical pathway 3 Al-Busaidi I.S. Clare G.C. Joyce L.R. et al. Presentation, Treatment and Long-Term Outcomes of a Multidisciplinary Acute Atrial Fibrillation Pathway: A 12-Month Follow-Up Study. Heart Lung Circ. 2022; 31: 216-223 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar in 2015 which involves a ‘rate-and-wait’ strategy where patients are managed with rate control agents followed by a review in a ward-based clinic within 24-72 hours (97% attend within this time frame). Over two-thirds (71%) of pathway patients spontaneously reverted to SR by clinic review. 3 Al-Busaidi I.S. Clare G.C. Joyce L.R. et al. Presentation, Treatment and Long-Term Outcomes of a Multidisciplinary Acute Atrial Fibrillation Pathway: A 12-Month Follow-Up Study. Heart Lung Circ. 2022; 31: 216-223 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar We examined predictors of spontaneous reversion to SR at AF clinic follow-up.

Nanoscale insights into the local structural rearrangements of amyloid-β induced by bexarotene.

A better understanding of the abnormal protein aggregation and the effect of anti-aggregation agents on the fibrillation pathways and the secondary structure of aggregates can determine strategies for the early treatment of dementia. Herein, we present a combination of experimental and theoretical studies providing new insights into the influence of the anti-aggregation drug bexarotene on the secondary structure of individual amyloid-β aggregates and its primary aggregation. The molecular rearrangements and the spatial distribution of β-sheets within individual aggregates were monitored at the nanoscale with infrared nanospectroscopy. We observed that bexarotene limits the parallel β-sheets formation, known to be highly abundant in fibrils at later phases of the amyloid-β aggregation composed of in-register cross-β structure. Moreover, we applied molecular dynamics to provide molecular-level insights into the investigated system. Both theoretical and experimental results revealed that bexarotene slows down the protein aggregation process via steric effects, largely prohibiting the antiparallel to parallel β-sheet rearrangement. We also found that bexarotene interacts not only via the single hydrogen bond formation with the peptide backbone but also with the amino acid side residue via a hydrophobic effect. The studied model of the drug-amyloid-β interaction contributes to a better understanding of the inhibition mechanism of the amyloid-β aggregation by the small molecule drugs. However, our nanoscale findings need to meet in vivo research requiring different analytical approaches.

The role of surface activity on the amyloid fibrillation pathway of bovine serum albumin upon interaction with glyphosate

As an active ingredient in its derivative products, glyphosate has emerged as the most widespread herbicide in recent decades. Bovine serum albumin (BSA) as a carrier protein may be adversely affected by structural changes due to binding affinity with glyphosate, which may lead to dysfunctionality or metabolic disorders. This study aimed to investigate the interaction of glyphosate with BSA and its thermal fibrillation pathway employing techniques such as dynamic surface tension, fluorescence quenching, ThT binding, circular dichroism spectroscopy, and reactive oxygen species (ROS) measurement, as well as molecular dynamics (MD) studies. The adsorption dynamic analysis suggested hydrophobic moiety at higher concentrations of glyphosate upon interaction with BSA. MD results suggested a slight fluctuation due to glyphosate interaction with protein molecules. The carboxy group presented in glyphosate made a hydrogen bond with the hydroxyl group of TYR147. The fluorescence quenching and diffusion studies approved BSA's increased unfolding and hydrophobicity resulting from glyphosate interaction, which would induce fibrillation/aggregation, according to our fibrillation kinetics data. The surface activity of glyphosate at higher concentrations and its approved involvement in structural changes of BSA through hydrogen bonding may raise concerns about its potential side effect on farm animals and the food cycle.

Concerted enhanced-sampling simulations to elucidate the helix-fibril transition pathway of intrinsically disordered α-Synuclein

Fibril formation of α-synuclein is linked with Parkinson's disease. The intrinsically disordered nature of α-syn provides extensive conformational plasticity and becomes difficult to characterize its transition pathway from native monomeric to disease-associated fibril form. We implemented different simulation methods such as steered dynamics-umbrella sampling, and replica-exchange and conventional MD simulations to access various conformational states of α-syn. Nineteen distinct intermediate structures were identified by free energy landscape and cluster analysis. They were then sorted based on secondary structure and solvent exposure of fibril-core residues to illustrate the fibril dissociation pathway. The analysis showed that following the initial dissociation of the polypeptide chain from the fibril, α-syn might form either compact-conformations by long-range interactions or extended-conformations stabilized by local interactions. This leads α-syn to adapt two different pathways. The secondary structure, solvation, contact distance, interaction energies and backbone dihedrals of thirty-two selected residues were analyzed for all the 19 intermediates. The results suggested that formation of β-turns, reorganization of salt bridges, and dihedral changes in the hydrophobic regions are the major driving forces for helix-fibril transition. Structural features of the intermediates also correlated with the earlier experimental and computational studies. The study provides critical information on the fibrillation pathway of α-syn.

Effect of CaCl2 on 2 heat-induced whey protein concentrate fibrillation pathways: Spontaneous and nuclear induction

Amyloid fibrils have many excellent functional properties that facilitate their applications in the food industry. There are 2 pathways for whey protein concentrate (WPC) to form amyloid fibril aggregates: spontaneous pathway and nuclear induction pathway. Low ionic strength is a necessary condition for the spontaneous pathway to proceed successfully. In this paper, the effect of salt ions on 2 WPC fibrillation pathways was investigated by adding CaCl2. The results demonstrated WPC fibrils were unable to form normally through spontaneous pathway as adding CaCl2; but still could form through nuclear induction pathway with 20 to 30 mM CaCl2, the nuclei accelerated the fibrillation process led to the resistance to the disordered aggregation brought by CaCl2. Moreover, divalent cations (Ca2+, Mg2+) had much stronger effects than monovalent cations (Na+) on fibril formation, and the results of X-ray photoelectron spectrum together with Fourier-transform infrared spectroscopy suggested that Ca2+ had a greater effect on the fibril formation than Cl-.

Development of a Care Pathway for Atrial Fibrillation Patients in the Emergency Department.

Atrial fibrillation (AF) is the most common cardiac arrhythmia and its prevalence is continuously increasing in the United States, leading to a progressive rise in the number of disease-related emergency department (ED) visits and hospitalizations. Although optimal long-term outpatient management for AF is well defined, the guidelines for optimal ED management of acute AF episodes is less clear. Studies have demonstrated that discharging patients with AF from the ED after acute stabilization is both safe and cost effective; however, the majority of these patients in the United States and in our institution are admitted to the hospital. To improve care of these patients, we established a multidisciplinary collaboration to develop an evidence-based systematic approach for the treatment and management of AF in the ED, that led to the creation of the University of California-Cardioversion, Anticoagulation, Rate Control, Expedited Follow-up/Education Atrial Fibrillation Pathway. Our pathway focuses on the acute stabilization of AF, adherence to best practices for anticoagulation, and reduction in unnecessary admissions through discharge from the ED with expedited outpatient follow-up whenever safe. A novel aspect of our pathway is that it is primarily driven by the ED physicians, while other published protocols primarily involve consulting cardiologists to guide management in the ED. Our protocol is very pertinent considering the current trend toward increased AF prevalence in the United States, coupled with a need for widespread implementation of strategies aimed at improving management of these patients while safely reducing hospital admissions and the economic burden of AF.

Probing the Influence of Single-Site Mutations in the Central Cross-β Region of Amyloid β (1-40) Peptides.

Amyloid β (Aβ) is a peptide known to form amyloid fibrils in the brain of patients suffering from Alzheimer’s disease. A complete mechanistic understanding how Aβ peptides form neurotoxic assemblies and how they kill neurons has not yet been achieved. Previous analysis of various Aβ40 mutants could reveal the significant importance of the hydrophobic contact between the residues Phe19 and Leu34 for cell toxicity. For some mutations at Phe19, toxicity was completely abolished. In the current study, we assessed if perturbations introduced by mutations in the direct proximity of the Phe19/Leu34 contact would have similar relevance for the fibrillation kinetics, structure, dynamics and toxicity of the Aβ assemblies. To this end, we rationally modified positions Phe20 or Gly33. A small library of Aβ40 peptides with Phe20 mutated to Lys, Tyr or the non-proteinogenic cyclohexylalanine (Cha) or Gly33 mutated to Ala was synthesized. We used electron microscopy, circular dichroism, X-ray diffraction, solid-state NMR spectroscopy, ThT fluorescence and MTT cell toxicity assays to comprehensively investigate the physicochemical properties of the Aβ fibrils formed by the modified peptides as well as toxicity to a neuronal cell line. Single mutations of either Phe20 or Gly33 led to relatively drastic alterations in the Aβ fibrillation kinetics but left the global, as well as the local structure, of the fibrils largely unchanged. Furthermore, the introduced perturbations caused a severe decrease or loss of cell toxicity compared to wildtype Aβ40. We suggest that perturbations at position Phe20 and Gly33 affect the fibrillation pathway of Aβ40 and, thereby, influence the especially toxic oligomeric species manifesting so that the region around the Phe19/Leu34 hydrophobic contact provides a promising site for the design of small molecules interfering with the Aβ fibrillation pathway.

Carvedilol inhibits Aβ25-35 fibrillation by intervening the early stage helical intermediate formation: A biophysical investigation

Self-assembly of disordered amyloid-beta (Aβ) peptides results in highly ordered amyloid fibrils. The structural information of the early-stage events and also in the presence of inhibitors is of great significance. It is challenging to acquire due to the nature of the amyloids and experimental constraints. Here, we demonstrate the cascade of aggregation (early to late) of the Aβ25-35 peptide in the absence and presence of carvedilol, a nonselective β-adrenergic receptor blocker. The aggregation process of Aβ25-35 peptide is monitored using Thioflavin T (ThT) fluorescence, dynamic light scattering (DLS), circular dichroism (CD), Raman spectroscopic techniques, and imaging experiments. We find that the Aβ25-35 peptide undergoes an early-stage (3-6 h) helical intermediate formation across the fibrillation pathway using CD and Raman measurements. Carvedilol obstructs the helical intermediate formation of Aβ25-35 peptide resulting in inhibition. CD spectra and deconvolution of the Raman bands suggest the β-sheet formation (24-100 h) in the absence of carvedilol. Spectroscopic results indicate a disordered structure for the peptide in the presence of carvedilol (24-100 h). Electron microscopy (EM) shows the formation of polymorphic fibrils for the peptide alone and non-amyloidal aggregates in the presence of carvedilol. Molecular docking study suggests that the plausible mode of interaction with carvedilol involves the C-terminal residues of the peptide.

Cover Feature: Remodeling of the Fibrillation Pathway of α‐Synuclein by Interaction with Antimicrobial Peptide LL‐III (Chem. Eur. J. 46/2021)

Liquid–liquid phase separation plus the droplet formation of proteins has emerged as a key mechanism for intracellular organization, but there is also evidence linking it to a variety of medical conditions. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via such droplet condensation. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, stabilizing the droplet phase and preventing conversion to the toxic fibrillar state. More information can be found in the Communication by R. Oliva, R. Winter, et al. (DOI: 10.1002/chem.202101592).

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III.

Liquid‐liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α‐synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL‐III is able to interact with both monomers and condensates of α‐synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti‐aggregation activity of LL‐III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial‐type peptides with liquid condensates such as α‐synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.

Presentation, Treatment and Long-Term Outcomes of a Multidisciplinary Acute Atrial Fibrillation Pathway: A 12-Month Follow-Up Study

Atrial fibrillation/flutter (AF/AFL) is associated with high rates of emergency department (ED) visits and acute hospitalisation. A recently established multidisciplinary acute AF treatment pathway seeks to avoid hospital admissions by early discharge of haemodynamically stable, low risk patients from the ED with next-working-day return to a ward-based AF clinic for further assessment. We conducted a preliminary analysis of the clinical outcomes of this pathway. We retrospectively reviewed clinical records of all patients assessed at the AF clinic at Christchurch Hospital, New Zealand, over a 12-month period. Data related to presentation, patient characteristics, treatment, and 12-month outcomes were analysed. A total of 143 patients (median age 65, interquartile range: 57-74 years, 59% male, 87% European) were assessed. Of these, 87 (60.8%) presented with their first episode of AF/AFL. Spontaneous cardioversion occurred in 41% at ED discharge, and this increased to 73% at AF clinic review. Electrical cardioversion was subsequently performed in 16 patients (11.2%), and 16 (11.2%) ultimately required hospital admission (eight to facilitate electrical cardioversion). At a median of 1 day, 83.9% were discharged from the AF clinic in sinus rhythm. During 12-month follow-up, there were 25 AF-related hospitalisations (20 patients, 14%) and one patient underwent electrical cardioversion; additionally, one patient had had a stroke and eight had bleeding complications giving a combined outcome rate of 6.3%. Utilising a rate-control strategy with ED discharge and early return to a dedicated AF clinic can safely prevent the majority of hospitalisations, avert unnecessary procedures, and facilitate longitudinal care.

Conformational distortion in a fibril-forming oligomer arrests alpha-Synuclein fibrillation and minimizes its toxic effects

The fibrillation pathway of alpha-Synuclein, the causative protein of Parkinson’s disease, encompasses transient, heterogeneous oligomeric forms whose structural understanding and link to toxicity are not yet understood. We report that the addition of the physiologically-available small molecule heme at a sub-stoichiometric ratio to either monomeric or aggregated α-Syn, targets a His50 residue critical for fibril-formation and stabilizes the structurally-heterogeneous populations of aggregates into a minimally-toxic oligomeric state. Cryo-EM 3D reconstruction revealed a ‘mace’-shaped structure of this monodisperse population of oligomers, which is comparable to a solid-state NMR Greek key-like motif (where the core residues are arranged in parallel in-register sheets with a Greek key topology at the C terminus) that forms the fundamental unit/kernel of protofilaments. Further structural analyses suggest that heme binding induces a distortion in the Greek key-like architecture of the mace oligomers, which impairs their further appending into protofilaments and fibrils. Additionally, our study reports a novel mechanism of prevention as well as reclamation of amyloid fibril formation by blocking an inter-protofilament His50 residue using a small molecule.

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.