Exploring the potential of deep-blue autofluorescence for monitoring amyloid fibril formation and dissociation

Protein aggregation into amyloid fibrils has been linked to multiple neurodegenerative disorders. Determining the kinetics of fibril formation, as well as their structural stability are important for the mechanistic understanding of amyloid aggregation. Tracking both fibril association and dissociation is usually performed by measuring light scattering of the solution or fluorescence of amyloid specific dyes, such as thioflavin-T. A possible addition to these methods is the recently discovered deep-blue autofluorescence (dbAF), which is linked to amyloid formation. In this work we explore the potential of this phenomenon to monitor amyloid fibril formation and dissociation, as well as show its possible relation to fibril size rather than amyloid structure.

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Identification of Novel 1,3,5-Triphenylbenzene Derivative Compounds as Inhibitors of Hen Lysozyme Amyloid Fibril Formation

International Journal of Molecular Sciences ◽

10.3390/ijms20225558 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5558

Author(s):

Hassan Ramshini ◽

Reza Tayebee ◽

Alessandra Bigi ◽

Francesco Bemporad ◽

Cristina Cecchi ◽

...

Keyword(s):

Amyloid Fibrils ◽

Amyloid Fibril ◽

Inhibitory Effect ◽

Fibril Formation ◽

Amyloid Formation ◽

Binding Assays ◽

Egg White Lysozyme ◽

Amyloid Fibril Formation ◽

Activity Measurements ◽

Model Protein

Deposition of soluble proteins as insoluble amyloid fibrils is associated with a number of pathological states. There is a growing interest in the identification of small molecules that can prevent proteins from undergoing amyloid fibril formation. In the present study, a series of small aromatic compounds with different substitutions of 1,3,5-triphenylbenzene have been synthesized and their possible effects on amyloid fibril formation by hen egg white lysozyme (HEWL), a model protein for amyloid formation, and of their resulting toxicity were examined. The inhibitory effect of the compounds against HEWL amyloid formation was analyzed using thioflavin T and Congo red binding assays, atomic force microscopy, Fourier-transform infrared spectroscopy, and cytotoxicity assays, such as the 3-(4,5-Dimethylthiazol)-2,5-Diphenyltetrazolium Bromide (MTT) reduction assay and caspase-3 activity measurements. We found that all compounds in our screen were efficient inhibitors of HEWL fibril formation and their associated toxicity. We showed that electron-withdrawing substituents such as –F and –NO2 potentiated the inhibitory potential of 1,3,5-triphenylbenzene, whereas electron-donating groups such as –OH, –OCH3, and –CH3 lowered it. These results may ultimately find applications in the development of potential inhibitors against amyloid fibril formation and its biologically adverse effects.

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Sensing and modulation of amyloid fibrils by photo-switchable organic dots

Nanoscale ◽

10.1039/d0nr04312e ◽

2020 ◽

Vol 12 (32) ◽

pp. 16805-16818

Author(s):

Aslam Uddin ◽

Bibhisan Roy ◽

Gregor P. Jose ◽

Sk Saddam Hossain ◽

Partha Hazra

Keyword(s):

Amyloid Fibrils ◽

Amyloid Fibril ◽

Early Stage ◽

Fibril Formation ◽

Amyloid Formation ◽

Amyloid Fibril Formation

Our study demonstrates that organic dots can be used for the imaging and early stage detection of amyloid fibril formation and the modulation of amyloid formation pathways.

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The Aggregation Conditions Define Whether EGCG is an Inhibitor or Enhancer of α-Synuclein Amyloid Fibril Formation

International Journal of Molecular Sciences ◽

10.3390/ijms21061995 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1995 ◽

Cited By ~ 1

Author(s):

Rebecca Sternke-Hoffmann ◽

Alessia Peduzzo ◽

Najoua Bolakhrif ◽

Rainer Haas ◽

Alexander K. Buell

Keyword(s):

Amyloid Fibrils ◽

Amyloid Fibril ◽

Epigallocatechin Gallate ◽

Small Region ◽

Fibril Formation ◽

Oxidation Products ◽

Relevant Parameter ◽

Amyloid Formation ◽

Experimental Conditions ◽

Amyloid Fibril Formation

The amyloid fibril formation by α -synuclein is a hallmark of various neurodegenerative disorders, most notably Parkinson’s disease. Epigallocatechin gallate (EGCG) has been reported to be an efficient inhibitor of amyloid formation by numerous proteins, among them α -synuclein. Here, we show that this applies only to a small region of the relevant parameter space, in particular to solution conditions where EGCG readily oxidizes, and we find that the oxidation product is a much more potent inhibitor compared to the unmodified EGCG. In addition to its inhibitory effects, EGCG and its oxidation products can under some conditions even accelerate α -synuclein amyloid fibril formation through facilitating its heterogeneous primary nucleation. Furthermore, we show through quantitative seeding experiments that, contrary to previous reports, EGCG is not able to re-model α -synuclein amyloid fibrils into seeding-incompetent structures. Taken together, our results paint a complex picture of EGCG as a compound that can under some conditions inhibit the amyloid fibril formation of α -synuclein, but the inhibitory action is not robust against various physiologically relevant changes in experimental conditions. Our results are important for the development of strategies to identify and characterize promising amyloid inhibitors.

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Abstract 312: Amyloids of Oxidized Apolipoprotein A-I Induce Amyloid Fibril Formation in Intact Apolipoprotein A-I

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.312 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Gary K Chan ◽

Andrzej Witkowski ◽

Giorgio Cavigiolio

Keyword(s):

Amyloid Fibrils ◽

Amyloid Fibril ◽

Fibril Formation ◽

Atherosclerotic Plaques ◽

Amyloid Formation ◽

Atherosclerotic Lesions ◽

Apolipoprotein A ◽

Amyloid Deposits ◽

Amyloid Fibril Formation ◽

Tht Fluorescence

Background: Amyloid deposition in atherosclerotic plaques increases with aging. Although a correlation between arterial amyloid deposits and cardiovascular events is yet to be established, the high incidence of amyloids associated with aortic intima and with atherosclerotic lesions indicates that amyloid deposits may contribute to atherosclerosis progression. Remarkably, apolipoprotein A-I (apoA-I) is the main component of these amyloids. We previously demonstrated that oxidation of apoA-I methionines by myeloperoxidase, at a concentration similar to that produced by activated macrophages in atherosclerotic lesions, promotes apoA-I amyloid fibril formation. Furthermore, recent studies revealed a hundred-fold increase in the amount of lipid-free apoA-I in atherosclerotic arteries compared to normal arteries. Notably, this apoA-I is heavily oxidized. Thus in the atherosclerotic plaques, high concentration of lipid-free apoA-I and an oxidative milieu are favorable conditions for apoA-I amyloid formation. Hypothesis: We tested the hypothesis that amyloid fibrils constituted of oxidized apoA-I can transfer the dysfunctional phenotype to intact apoA-I. Methods: Pre-formed amyloid fibrils constituted of oxidized apoA-I were incubated with a 10-fold excess of intact apoA-I at 37 °C, pH 6.0, with continuous vortexing. Kinetics of amyloid fibril formation by the pool of intact apoA-I were derived by measuring Thioflavin-T (ThT) fluorescence over a 6-day period. Results: After a lag-phase of 24-48 h, fibril formation proceeded with typical sigmoidal kinetics and reached plateau levels after about 6 days. In control samples, in which intact apoA-I was incubated in the absence of pre-formed fibrils, no significant changes in ThT fluorescence were detected for the same time course. Conclusions: Oxidized apoA-I amyloid fibrils can catalyze the aggregation of a large excess of intact protein. This observation bears important pathophysiological implications. In vivo , a small amount of amyloid fibrils could be produced by oxidized apoA-I in specific microenvironments of the atheroma; when transferred to the surrounding tissues, these amyloid seeds could induce extended amyloid formation in the large available pool of lipid-free apoA-I.

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New Mechanism of Amyloid Fibril Formation

Current Protein and Peptide Science ◽

10.2174/1389203720666190125160937 ◽

2019 ◽

Vol 20 (6) ◽

pp. 630-640 ◽

Cited By ~ 10

Author(s):

Oxana Galzitskaya

Keyword(s):

Amyloid Fibrils ◽

Amyloid Fibril ◽

Molecular Model ◽

Fibril Formation ◽

Mass Spectrometry Data ◽

Amyloid Formation ◽

Aβ Peptides ◽

Amyloid Fibril Formation ◽

A Cell ◽

Amyloid Structures

Polymorphism is a specific feature of the amyloid structures. We have studied the amyloid structures and the process of their formation using the synthetic and recombinant preparations of Aβ peptides and their three fragments. The fibrils of different morphology were obtained for these peptides. We suppose that fibril formation by Aβ peptides and their fragments proceeds according to the simplified scheme: destabilized monomer → ring-like oligomer → mature fibril that consists of ringlike oligomers. We are the first who did 2D reconstruction of amyloid fibrils provided that just a ringlike oligomer is the main building block in fibril of any morphology, like a cell in an organism. Taking this into account it is easy to explain the polymorphism of fibrils as well as the splitting of mature fibrils under different external actions, the branching and inhomogeneity of fibril diameters. Identification of regions in the protein chains that form the backbone of amyloid fibril is a direction in the investigation of amyloid formation. It has been demonstrated for Aβ(1-42) peptide and its fragments that their complete structure is inaccessible for the action of proteases, which is an evidence of different ways of association of ring-like oligomers with the formation of fibrils. Based on the electron microscopy and mass spectrometry data, we have proposed a molecular model of the fibril formed by both Aβ peptide and its fragments. In connection with this, the unified way of formation of fibrils by oligomers, which we have discovered, could facilitate the development of relevant fields of medicine of common action.

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IS IT POSSIBLE TO PREDICT AMYLOIDOGENIC REGIONS FROM SEQUENCE ALONE?

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720006002004 ◽

2006 ◽

Vol 04 (02) ◽

pp. 373-388 ◽

Cited By ~ 19

Author(s):

OXANA V. GALZITSKAYA ◽

SERGIY O. GARBUZYNSKIY ◽

MICHAIL YU. LOBANOV

Keyword(s):

Amino Acid ◽

Protein Sequence ◽

Amyloid Fibrils ◽

Amyloid Fibril ◽

Fibril Formation ◽

Amino Acid Sequences ◽

Amyloid Formation ◽

Amyloid Fibril Formation ◽

Polypeptide Chains ◽

Proteins And Peptides

Identification of potentially amyloidogenic regions in polypeptide chains is very important because the amyloid fibril formation can be induced in most normal proteins. In our work we suggest a new method to detect amyloidogenic regions in protein sequence. It is based on the assumption that packing is tight inside an amyloid and therefore regions which could potentially pack well would have a tendency to form amyloids. This means that the regions with strong expected packing of residues would be responsible for the amyloid formation. We use this property to identify potentially amyloidogenic regions in proteins basing on their amino acid sequences only. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Predictions of the regions which are responsible for the formation of amyloid fibrils in proteins unrelated to disease have been also done.

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The amphibian antimicrobial peptide uperin 3.5 is a cross-α/cross-β chameleon functional amyloid

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014442118 ◽

2021 ◽

Vol 118 (3) ◽

pp. e2014442118

Author(s):

Nir Salinas ◽

Einav Tayeb-Fligelman ◽

Massimo D. Sammito ◽

Daniel Bloch ◽

Raz Jelinek ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Amyloid Fibrils ◽

Amyloid Fibril ◽

Membrane Damage ◽

Fibril Formation ◽

Regulation Mechanism ◽

Bacterial Cells ◽

Amyloid Fibril Formation ◽

Β Amyloid ◽

Β Sheets

Antimicrobial activity is being increasingly linked to amyloid fibril formation, suggesting physiological roles for some human amyloids, which have historically been viewed as strictly pathological agents. This work reports on formation of functional cross-α amyloid fibrils of the amphibian antimicrobial peptide uperin 3.5 at atomic resolution, an architecture initially discovered in the bacterial PSMα3 cytotoxin. The fibrils of uperin 3.5 and PSMα3 comprised antiparallel and parallel helical sheets, respectively, recapitulating properties of β-sheets. Uperin 3.5 demonstrated chameleon properties of a secondary structure switch, forming mostly cross-β fibrils in the absence of lipids. Uperin 3.5 helical fibril formation was largely induced by, and formed on, bacterial cells or membrane mimetics, and led to membrane damage and cell death. These findings suggest a regulation mechanism, which includes storage of inactive peptides as well as environmentally induced activation of uperin 3.5, via chameleon cross-α/β amyloid fibrils.

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What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

Journal of Biological Chemistry ◽

10.1074/jbc.m115.699348 ◽

2015 ◽

Vol 291 (4) ◽

pp. 2018-2032 ◽

Cited By ~ 29

Author(s):

Rosa Crespo ◽

Eva Villar-Alvarez ◽

Pablo Taboada ◽

Fernando A. Rocha ◽

Ana M. Damas ◽

...

Keyword(s):

Amyloid Fibrils ◽

Amyloid Fibril ◽

Pathogenic Role ◽

Amyloid Deposits ◽

Amyloid Fibril Formation ◽

Microscopy Techniques ◽

Analytical Tools ◽

Kinetics Of ◽

Quantitative Importance

Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. Although the pathogenic role of these fibrils has not been completely established, increasing evidence suggests off-pathway aggregation as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step toward the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of off-pathway aggregation relative to amyloid fibrillization; these include non-linear semilog plots of amyloid progress curves, highly variable end point signals, and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of dynamic light scattering, CD, and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery.

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