Co-Aggregation of S100A9 with DOPA and Cyclen-Based Compounds Manifested in Amyloid Fibril Thickening without Altering Rates of Self-Assembly

The amyloid cascade is central for the neurodegeneration disease pathology, including Alzheimer’s and Parkinson’s, and remains the focus of much current research. S100A9 protein drives the amyloid-neuroinflammatory cascade in these diseases. DOPA and cyclen-based compounds were used as amyloid modifiers and inhibitors previously, and DOPA is also used as a precursor of dopamine in Parkinson’s treatment. Here, by using fluorescence titration experiments we showed that five selected ligands: DOPA-D-H-DOPA, DOPA-H-H-DOPA, DOPA-D-H, DOPA-cyclen, and H-E-cyclen, bind to S100A9 with apparent Kd in the sub-micromolar range. Ligand docking and molecular dynamic simulation showed that all compounds bind to S100A9 in more than one binding site and with different ligand mobility and H-bonds involved in each site, which all together is consistent with the apparent binding determined in fluorescence experiments. By using amyloid kinetic analysis, monitored by thioflavin-T fluorescence, and AFM imaging, we found that S100A9 co-aggregation with these compounds does not hinder amyloid formation but leads to morphological changes in the amyloid fibrils, manifested in fibril thickening. Thicker fibrils were not observed upon fibrillation of S100A9 alone and may influence the amyloid tissue propagation and modulate S100A9 amyloid assembly as part of the amyloid-neuroinflammatory cascade in neurodegenerative diseases.

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Quantification of amyloid fibril polymorphism by nano-morphometry reveals the individuality of filament assembly

Communications Chemistry ◽

10.1038/s42004-020-00372-3 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 1

Author(s):

Liam D. Aubrey ◽

Ben J. F. Blakeman ◽

Liisa Lutter ◽

Christopher J. Serpell ◽

Mick F. Tuite ◽

...

Keyword(s):

Self Assembly ◽

Amyloid Fibrils ◽

Amyloid Fibril ◽

Biological Function ◽

Structural Basis ◽

Distance Curve ◽

Filament Assembly ◽

Sample Heterogeneity ◽

Structural Insights ◽

Polymorphic Nature

Abstract Amyloid fibrils are highly polymorphic structures formed by many different proteins. They provide biological function but also abnormally accumulate in numerous human diseases. The physicochemical principles of amyloid polymorphism are not understood due to lack of structural insights at the single-fibril level. To identify and classify different fibril polymorphs and to quantify the level of heterogeneity is essential to decipher the precise links between amyloid structures and their functional and disease associated properties such as toxicity, strains, propagation and spreading. Employing gentle, force-distance curve-based AFM, we produce detailed images, from which the 3D reconstruction of individual filaments in heterogeneous amyloid samples is achieved. Distinctive fibril polymorphs are then classified by hierarchical clustering, and sample heterogeneity is objectively quantified. These data demonstrate the polymorphic nature of fibril populations, provide important information regarding the energy landscape of amyloid self-assembly, and offer quantitative insights into the structural basis of polymorphism in amyloid populations.

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Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421204112 ◽

2015 ◽

Vol 112 (16) ◽

pp. E1994-E2003 ◽

Cited By ~ 228

Author(s):

Serene W. Chen ◽

Srdja Drakulic ◽

Emma Deas ◽

Myriam Ouberai ◽

Francesco A. Aprile ◽

...

Keyword(s):

Structural Characterization ◽

Self Assembly ◽

Protein Misfolding ◽

Amyloid Fibril ◽

Amyloid Formation ◽

Image Reconstruction Techniques ◽

Cryo Electron Microscopy ◽

Amyloid Oligomers ◽

Β Sheet

We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.

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Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013012 ◽

2020 ◽

Vol 295 (21) ◽

pp. 7544-7553 ◽

Cited By ~ 1

Author(s):

Dexter N. Dean ◽

Jennifer C. Lee

Keyword(s):

Alternative Splicing ◽

Amyloid Fibrils ◽

Limited Proteolysis ◽

Amyloid Formation ◽

Functional Amyloid ◽

Melanin Biosynthesis ◽

Functional Amyloids ◽

Fibril Morphology ◽

Amyloid Assembly ◽

Β Sheet

The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Multiple PMEL17 isoforms are produced, two of which arise from excision of a cryptic intron within the amyloid-forming repeat (RPT) domain, leading to long (lRPT) and short (sRPT) isoforms with 10 and 7 imperfect repeats, respectively. Both lRPT and sRPT isoforms undergo similar pH-dependent mechanisms of amyloid formation and fibril dissolution. Here, using human PMEL17, we tested the hypothesis that the minor, but more aggregation-prone, sRPT facilitates amyloid formation of lRPT. We observed that cross-seeding by sRPT fibrils accelerates the rate of lRPT aggregation, resulting in propagation of an sRPT-like twisted fibril morphology, unlike the rodlike structure that lRPT normally adopts. This templating was specific, as the reversed reaction inhibited sRPT fibril formation. Despite displaying ultrastructural differences, self- and cross-seeded lRPT fibrils had a similar β-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a “nucleator” of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.

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Structure and Aggregation Mechanisms in Amyloids

Molecules ◽

10.3390/molecules25051195 ◽

2020 ◽

Vol 25 (5) ◽

pp. 1195 ◽

Cited By ~ 6

Author(s):

Zaida L. Almeida ◽

Rui M. M. Brito

Keyword(s):

Molecular Species ◽

Molecular Mechanisms ◽

Amyloid Fibrils ◽

Current Knowledge ◽

Point Of View ◽

Resonance Spectroscopy ◽

Amyloid Formation ◽

Amyloid Fibril Formation ◽

Misfolding Diseases ◽

Amyloid Cascade

The aggregation of a polypeptide chain into amyloid fibrils and their accumulation and deposition into insoluble plaques and intracellular inclusions is the hallmark of several misfolding diseases known as amyloidoses. Alzheimer′s, Parkinson′s and Huntington’s diseases are some of the approximately 50 amyloid diseases described to date. The identification and characterization of the molecular species critical for amyloid formation and disease development have been the focus of intense scrutiny. Methods such as X-ray and electron diffraction, solid-state nuclear magnetic resonance spectroscopy (ssNMR) and cryo-electron microscopy (cryo-EM) have been extensively used and they have contributed to shed a new light onto the structure of amyloid, revealing a multiplicity of polymorphic structures that generally fit the cross-β amyloid motif. The development of rational therapeutic approaches against these debilitating and increasingly frequent misfolding diseases requires a thorough understanding of the molecular mechanisms underlying the amyloid cascade. Here, we review the current knowledge on amyloid fibril formation for several proteins and peptides from a kinetic and thermodynamic point of view, the structure of the molecular species involved in the amyloidogenic process, and the origin of their cytotoxicity.

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The RHIM within the M45 protein from murine cytomegalovirus forms heteromeric amyloid fibrils with RIPK1 and RIPK3

10.1101/324590 ◽

2018 ◽

Author(s):

Chi L. L. Pham ◽

Merryn Strange ◽

Ailis O’ Carroll ◽

Nirukshan Shanmugam ◽

Emma Sierecki ◽

...

Keyword(s):

Cell Death ◽

Biological Activity ◽

Self Assembly ◽

Amyloid Fibrils ◽

Human Cells ◽

Terminal Region ◽

Murine Cytomegalovirus ◽

Homotypic Interaction ◽

Amyloid Assembly

AbstractThe M45 protein from murine cytomegalovirus protects infected murine cells from death by necroptosis and can protect human cells from necroptosis induced by TNFR activation, when heterologously expressed. We show that the N-terminal 90 residues of the M45 protein, which contain a RIP Homotypic Interaction Motif (RHIM), are sufficient to confer protection against TNFR-induced necroptosis. This N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils and interacts with the RHIMs of human RIPK1 and RIPK3 kinases to form heteromeric amyloid fibrils in vitro. An intact RHIM core tetrad is required for the inhibition of cell death by M45 and we show that mutation of those key tetrad residues abolishes homo- and hetero-amyloid assembly by M45 in vitro, suggesting that the amyloidogenic nature of the M45 RHIM underlies its biological activity. Our results indicate that M45 mimics the interactions made by RIPK1 with RIPK3 in forming heteromeric amyloid structures.

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Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules

10.26434/chemrxiv.13008212 ◽

2020 ◽

Author(s):

Emma Cawood ◽

Nicolas Guthertz ◽

Jessica Ebo ◽

Theodoros Karamanos ◽

Sheena E. Radford FRS ◽

...

Keyword(s):

Protein Interactions ◽

Structural Characterization ◽

Self Assembly ◽

Molecular Mechanisms ◽

Amyloid Fibrils ◽

Fibril Formation ◽

X Ray Crystallography ◽

Starting Point ◽

Amyloid Assembly

Protein-protein interactions (PPIs) are involved in many of life’s essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific intermediates for detailed study. Such information can also provide a starting point for drug discovery. Here, we demonstrate that covalently tethered small molecule fragments can be used to stabilize specific oligomers during amyloid fibril formation, facilitating the structural characterization of these assembly intermediates. We exemplify the power of covalent tethering using the naturally occurring truncated variant (ΔN6) of the human protein β2-microglobulin (β2m), which assembles into amyloid fibrils associated with dialysis-related amyloidosis. Using this approach, we have trapped tetramers formed by ΔN6 under conditions which would normally lead to fibril formation and found that the degree of tetramer stabilization depends on the site of the covalent tether and the nature of the protein-fragment interaction. The covalent protein-ligand linkage enabled structural characterization of these trapped oligomeric species using X-ray crystallography and NMR, providing insight into why tetramer stabilization inhibits amyloid assembly. Our findings highlight the power of “post-translational chemical modification" as a tool to study biological molecular mechanisms.

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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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Aß40 displays amyloidogenic properties in the non-transgenic mouse brain but does not exacerbate Aß42 toxicity in Drosophila.

10.21203/rs.3.rs-47473/v3 ◽

2020 ◽

Author(s):

Lorena de Mena ◽

Michael A Smith ◽

Jason Martin ◽

Katie L Dunton ◽

Carolina Ceballos-Diaz ◽

...

Keyword(s):

Mouse Brain ◽

Self Assembly ◽

Amyloid Fibrils ◽

Amyloid Β ◽

Model Systems ◽

Amyloid Angiopathy ◽

Amyloid Deposits ◽

Amyloid Aggregates ◽

Amyloid Cascade

Abstract Background Self-assembly of the amyloid-β (Aβ) peptide into aggregates, from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer’s disease (AD). However it is clear that not all forms of Aβ are equally harmful, and that linking a specific aggregate to toxicity also depends on the assays and model systems used [1, 2]. Though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aβ deposition and neurodegeneration. Methods In this study, we examined familial mutations of Aβ that increase aggregation and oligomerization, E22G and DE22, and induce cerebral amyloid angiopathy, E22Q and D23N. We also investigated synthetic mutations that stabilize dimerization, S26C, and a phospho-mimetic, S8E, and non-phospho-mimetic, S8A. To that end, we utilized BRI2-Aβ fusion technology and rAAV2/1 based somatic brain transgenesis in mice to selectively express individual mutant Aβ species in vivo. In parallel we generated PhiC31-based transgenic Drosophila melanogaster expressing wild type (WT) and Aβ40 and Aβ42 mutants, fused to the Argos signal peptide to assess the extent of Aβ42-induced toxicity as well as to interrogate the combined effect of different Aβ40 and Aβ42 species.Results When expressed in the mouse brain for 6 months, Aβ42 E22G, Aβ42 E22Q/D23N, and Aβ42WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aβ40WT showed no distinctive phenotype, Aβ40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aβ40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aβ42 E22G, E22Q, and S26C, but not Aβ40, were toxic to the eye of Drosophila. In contrast, flies expressing a copy of Aβ40 (WT or mutants) in addition to Aβ42WT, showed improved phenotypes, suggesting possible protective qualities for Aβ40. Conclusions These studies suggest that while some Aβ40 mutants form unique amyloid aggregates in mouse brains, they do not exacerbate Aβ42 toxicity in Drosophila, which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.

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Thermodynamics of Amyloid Fibril Formation from Chemical Depolymerization

10.26434/chemrxiv.9632153.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Nicola Vettore ◽

Alexander Buell

Keyword(s):

Ionic Strength ◽

Thermodynamic Stability ◽

Amyloid Fibrils ◽

Amyloid Fibril ◽

Polymerization Process ◽

Published Data ◽

Amyloid Formation ◽

Ionic Strength Dependence ◽

Mechanisms Of Formation

Amyloid fibrils are homo-molecular protein polymers that play an important role in disease and biological function. While much is known about their kinetics and mechanisms of formation, the origin and magnitude of their thermodynamic stability has received significantly less attention. This is despite the fact that the thermodynamic stability of amyloid fibrils is an important determinant of their lifetimes and processing in vivo. Here we use depolymerization by chemical denaturants of amyloid fibrils of two different proteins (PI3K-SH3 and glucagon) at different concentrations and show that the previously applied linear polymerization model is an oversimplification that does not capture the concentration dependence of chemical depolymerization of amyloid fibrils. We show that cooperative polymerization, which is compatible with the picture of amyloid formation as a nucleated polymerization process, is able to quantitatively describe the thermodynamic data. We use this combined experimental and conceptual framework in order to probe the ionic strength dependence of amyloid fibril stability. In combination with previously published data on the ionic strength dependence of amyloid fibril growth kinetics, our results provide strong evidence for the product-like nature of the transition state of amyloid fibril growth.

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