scholarly journals Kinetic Transition in Amyloid Assembly as a Screening Assay for Oligomer-Selective Dyes

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 539 ◽  
Author(s):  
Jeremy Barton ◽  
D. Sebastian Arias ◽  
Chamani Niyangoda ◽  
Gustavo Borjas ◽  
Nathan Le ◽  
...  
Keyword(s):  
Crystal Violet ◽  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Fibril Formation ◽  
Thioflavin T ◽  
Screening Assay ◽  
Kinetic Transition ◽  
Amyloid Assembly

Assembly of amyloid fibrils and small globular oligomers is associated with a significant number of human disorders that include Alzheimer’s disease, senile systemic amyloidosis, and type II diabetes. Recent findings implicate small amyloid oligomers as the dominant aggregate species mediating the toxic effects in these disorders. However, validation of this hypothesis has been hampered by the dearth of experimental techniques to detect, quantify, and discriminate oligomeric intermediates from late-stage fibrils, in vitro and in vivo. We have shown that the onset of significant oligomer formation is associated with a transition in thioflavin T kinetics from sigmoidal to biphasic kinetics. Here we showed that this transition can be exploited for screening fluorophores for preferential responses to oligomer over fibril formation. This assay identified crystal violet as a strongly selective oligomer-indicator dye for lysozyme. Simultaneous recordings of amyloid kinetics with thioflavin T and crystal violet enabled us to separate the combined signals into their underlying oligomeric and fibrillar components. We provided further evidence that this screening assay could be extended to amyloid-β peptides under physiological conditions. Identification of oligomer-selective dyes not only holds the promise of biomedical applications but provides new approaches for unraveling the mechanisms underlying oligomer versus fibril formation in amyloid assembly.

scholarly journals The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

Molecules ◽  
2021 ◽  
Vol 26 (20) ◽  
pp. 6120
Author(s):  
Danielle M. Williams ◽  
David C. Thorn ◽  
Christopher M. Dobson ◽  
Sarah Meehan ◽  
Sophie E. Jackson ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Molecular Chaperones ◽  
Protein Unfolding ◽  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Parkinson’S Diseases ◽  
Amino Acid Form ◽  
Shock Proteins

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aβ40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

scholarly journals Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils

2019 ◽  
Vol 32 (2) ◽  
pp. 77-85
Author(s):  
Mohammad Ashhar I Khan ◽  
Ulrich Weininger ◽  
Sven Kjellström ◽  
Shashank Deep ◽  
Mikael Akke
Keyword(s):  
Superoxide Dismutase ◽  
Surface Area ◽  
Amyloid Fibrils ◽  
Hydrophobic Surface ◽  
Fibril Formation ◽  
Hydrophobic Surfaces ◽  
Molecular Features ◽  
Denaturant Concentration

Abstract Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.

An Aβ42 variant that inhibits intra- and extracellular amyloid aggregation and enhances cell viability

2018 ◽  
Vol 475 (19) ◽  
pp. 3087-3103 ◽  
Author(s):  
Ofek Oren ◽  
Victor Banerjee ◽  
Ran Taube ◽  
Niv Papo
Keyword(s):  
Amyloid Fibrils ◽  
Neuronal Cells ◽  
Amyloid Β ◽  
Neuronal Cell ◽  
Amyloid Β Peptide ◽  
Cell Cytotoxicity ◽  
Amyloid Aggregation ◽  
Molar Ratios

Aggregation and accumulation of the 42-residue amyloid β peptide (Aβ42) in the extracellular matrix and within neuronal cells is considered a major cause of neuronal cell cytotoxicity and death in Alzheimer's disease (AD) patients. Therefore, molecules that bind to Aβ42 and prevent its aggregation are therapeutically promising as AD treatment. Here, we show that a non-self-aggregating Aβ42 variant carrying two surface mutations, F19S and L34P (Aβ42DM), inhibits wild-type Aβ42 aggregation and significantly reduces Aβ42-mediated cell cytotoxicity. In addition, Aβ42DM inhibits the uptake and internalization of extracellularly added pre-formed Aβ42 aggregates into cells. This was the case in both neuronal and non-neuronal cells co-expressing Aβ42 and Aβ42DM or following pre-treatment of cells with extracellular soluble forms of the two peptides, even at high Aβ42 to Aβ42DM molar ratios. In cells, Aβ42DM associates with Aβ42, while in vitro, the two soluble recombinant peptides exhibit nano-molar binding affinity. Importantly, Aβ42DM potently suppresses Aβ42 amyloid aggregation in vitro, as demonstrated by thioflavin T fluorescence and transmission electron microscopy for detecting amyloid fibrils. Overall, we present a new approach for inhibiting Aβ42 fibril formation both within and outside cells. Accordingly, Aβ42DM should be evaluated in vivo for potential use as a therapeutic lead for treating AD.

scholarly journals Doxycycline reduces fibril formation in a transgenic mouse model of AL amyloidosis

Blood ◽  
2011 ◽  
Vol 118 (25) ◽  
pp. 6610-6617 ◽  
Author(s):  
Jennifer Ellis Ward ◽  
Ruiyi Ren ◽  
Gianluca Toraldo ◽  
Pam SooHoo ◽  
Jian Guan ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Amyloid Fibrils ◽  
Cell Clone ◽  
Ex Vivo ◽  
Organ Damage ◽  
Fibril Formation ◽  
Al Amyloidosis ◽  
Cmv Promoter

AbstractSystemic AL amyloidosis results from the aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) usually produced by a plasma cell clone in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as amyloid fibrils can rapidly accumulate in tissues including the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. Chemotherapy is used to eradicate the cellular source of the amyloidogenic precursor. Currently, there are no therapies that target the process of LC aggregation, fibril formation, or organ damage. We developed transgenic mice expressing an amyloidogenic λ6 LC using the cytomegalovirus (CMV) promoter to circumvent the disruption of B cell development by premature expression of recombined LC. The CMV-λ6 transgenic mice develop neurologic dysfunction and Congophilic amyloid deposits in the stomach. Amyloid deposition was inhibited in vivo by the antibiotic doxycycline. In vitro studies demonstrated that doxycycline directly disrupted the formation of recombinant LC fibrils. Furthermore, treatment of ex vivo LC amyloid fibrils with doxycycline reduced the number of intact fibrils and led to the formation of large disordered aggregates. The CMV-λ6 transgenic model replicates the process of AL amyloidosis and is useful for testing the antifibril potential of orally available agents.

scholarly journals Modulators of amyloid protein aggregation and toxicity: EGCG and CLR01

2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Aida Attar ◽  
Farid Rahimi ◽  
Gal Bitan
Keyword(s):  
Protein Aggregation ◽  
Drug Targets ◽  
Amyloid Fibrils ◽  
Specific Binding ◽  
Amyloid Β ◽  
Amyloid Β Protein ◽  
Amyloid Protein ◽  
Islet Amyloid

AbstractAbnormal protein folding and self-assembly causes over 30 cureless human diseases for which no disease-modifying therapies are available. The common side to all these diseases is formation of aberrant toxic protein oligomers and amyloid fibrils. Both types of assemblies are drug targets, yet each presents major challenges to drug design, discovery, and development. In this review, we focus on two small molecules that inhibit formation of toxic amyloid protein assemblies — the green-tea derivative (−)-epigallocatechin-3-gallate (EGCG), which was identified through a combination of epidemiologic data and a compound library screen, and the molecular tweezer CLR01, whose inhibitory activity was discovered in our group based on rational reasoning, and subsequently confirmed experimentally. Both compounds act in a manner that is not specific to one particular protein and thus are useful against a multitude of amyloidogenic proteins, yet they act via distinct putative mechanisms. CLR01 disrupts protein aggregation through specific binding to lysine residues, whereas the mechanisms underlying the activity of EGCG are only recently beginning to unveil. We discuss current in vitro and, where available, in vivo literature related to EGCG and CLR01’s effects on amyloid β-protein, α-synuclein, transthyretin, islet amyloid polypeptide, and calcitonin. We also describe the toxicity, pharmacokinetics, and mechanism of action of each compound.

scholarly journals C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice

2015 ◽  
Vol 112 (8) ◽  
pp. E836-E845 ◽  
Author(s):  
Jinko Sawashita ◽  
Beiru Zhang ◽  
Kazuhiro Hasegawa ◽  
Masayuki Mori ◽  
Hironobu Naiki ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Fibril Formation ◽  
Mouse Strains ◽  
Strong Inhibitor ◽  
Apolipoprotein A ◽  
Inhibitory Mechanisms ◽  
Amyloid Fibril Formation ◽  
Type C

In murine senile amyloidosis, misfolded serum apolipoprotein (apo) A-II deposits as amyloid fibrils (AApoAII) in a process associated with aging. Mouse strains carrying type C apoA-II (APOA2C) protein exhibit a high incidence of severe systemic amyloidosis. Previously, we showed that N- and C-terminal sequences of apoA-II protein are critical for polymerization into amyloid fibrils in vitro. Here, we demonstrate that congenic mouse strains carrying type F apoA-II (APOA2F) protein, which contains four amino acid substitutions in the amyloidogenic regions of APOA2C, were absolutely resistant to amyloidosis, even after induction of amyloidosis by injection of AApoAII. In vitro fibril formation tests showed that N- and C-terminal APOA2F peptides did not polymerize into amyloid fibrils. Moreover, a C-terminal APOA2F peptide was a strong inhibitor of nucleation and extension of amyloid fibrils during polymerization. Importantly, after the induction of amyloidosis, we succeeded in suppressing amyloid deposition in senile amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We suggest that the C-terminal APOA2F peptide might inhibit further extension of amyloid fibrils by blocking the active ends of nuclei (seeds). We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies.

Phenolic Acids Exert Anticholinesterase and Cognition-Improving Effects

2018 ◽  
Vol 15 (6) ◽  
pp. 531-543 ◽  
Author(s):  
Dominik Szwajgier ◽  
Ewa Baranowska-Wojcik ◽  
Kamila Borowiec
Keyword(s):  
Phenolic Acids ◽  
Ex Vivo ◽  
Amyloid Β ◽  
Inhibitory Effect ◽  
In Vivo Studies ◽  
Amyloid Β Peptide ◽  
Beneficial Effects ◽  
Aβ Fibrils

Numerous authors have provided evidence regarding the beneficial effects of phenolic acids and their derivatives against Alzheimer's disease (AD). In this review, the role of phenolic acids as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is discussed, including the structure-activity relationship. In addition, the inhibitory effect of phenolic acids on the formation of amyloid β-peptide (Aβ) fibrils is presented. We also cover the in vitro, ex vivo, and in vivo studies concerning the prevention and treatment of the cognitive enhancement.

scholarly journals Borrelidin from Saltern-Derived Halophilic Nocardiopsis sp. Dissociates Amyloid-β and Tau Fibrils

2020 ◽  
pp. 1-7
Author(s):  
Jisu Shin ◽  
Seung-Hoon Yang ◽  
Young Eun Du ◽  
Keunwan Park ◽  
DaWon Kim ◽  
...  
Keyword(s):  
Amyloid Β ◽  
Thioflavin T ◽  
Drug Candidate ◽  
Ht22 Cells ◽  
Aβ Aggregation ◽  
Actinomycete Strain ◽  
Direct Dissociation ◽  
Aβ Fibrils ◽  
Tau Aggregation

Background: Alzheimer’s disease (AD) is characterized by the aggregation of two pathological proteins, amyloid-β (Aβ) and tau, leading to neuronal and cognitive dysfunction. Clearance of either Aβ or tau aggregates by immunotherapy has become a potential therapy, as these aggregates are found in the brain ahead of the symptom onset. Given that Aβ and tau independently and cooperatively play critical roles in AD development, AD treatments might require therapeutic approaches to eliminate both aggregates together. Objective: We aimed to discover a chemical drug candidate from natural sources for direct dissociation of both insoluble Aβ and tau aggregates through in vitro assessments. Methods: We isolated four borrelidin chemicals from a saltern-derived halophilic actinomycete strain of rare genus Nocardiopsis and simulated their docking interactions with Aβ fibrils. Then, anti-cytotoxic, anti-Aβ, and anti-tau effects of borrelidins were examined by MTT assays with HT22 hippocampal cell line, thioflavin T assays, and gel electrophoresis. Results: When HT22 cells were exposed to Aβ aggregates, the treatment of borrelidins alleviates the Aβ-induced toxicity. These anti-cytotoxic effects can be derived from the inhibitory functions of borrelidins against the Aβ aggregation as shown in thioflavin T and gel electrophoretic analyses. Among them, especially borrelidin, which exhibits the highest probability of docking, not only dissociates Aβ aggregates but also directly regulates tau aggregation. Conclusion: Borrelidin dissociates insoluble Aβ and tau aggregates together and our findings support the view that it is possible to develop an alternative chemical approach mimicking anti-Aβ or anti-tau immunotherapy for clearance of both aggregates.

Neuroprotective Effects of the FGF21 Analogue LY2405319

2021 ◽  
pp. 1-13
Author(s):  
Claire Rühlmann ◽  
David Dannehl ◽  
Marcus Brodtrück ◽  
Andrew C. Adams ◽  
Jan Stenzel ◽  
...  
Keyword(s):  
Glial Cells ◽  
Neuroprotective Effect ◽  
Amyloid Β ◽  
Fibroblast Growth Factor 21 ◽  
Neuroprotective Effects ◽  
Organotypic Brain Slice ◽  
Brain Slice Cultures ◽  
Abca1 Mrna

Background: To date, there are no effective treatments for Alzheimer’s disease (AD). Thus, a significant need for research of therapies remains. Objective: One promising pharmacological target is the hormone fibroblast growth factor 21 (FGF21), which is thought to be neuroprotective. A clinical candidate for medical use could be the FGF21 analogue LY2405319 (LY), which has a specificity and potency comparable to FGF21. Methods: The present study investigated the potential neuroprotective effect of LY via PPARγ/apoE/abca1 pathway which is known to degrade amyloid-β (Aβ) plaques by using primary glial cells and hippocampal organotypic brain slice cultures (OBSCs) from 30- and 50-week-old transgenic APPswe/PS1dE9 (tg) mice. By LY treatment of 52-week-old tg mice with advanced Aβ deposition, we further aimed to elaborate the effect of LY on AD pathology in vivo. Results: LY application to primary glial cells caused an upregulation of pparγ, apoE, and abca1 mRNA expression and significantly decreased number and area of Aβ plaques in OBSCs. LY treatment in tg mice increased cerebral [18F] FDG uptake and N-acetylaspartate/creatine ratio indicating enhanced neuronal activity and integrity. Although LY did not reduce the number of Aβ plaques in tg mice, the number of iba1-positive cells was significantly decreased indicating reduced microgliosis. Conclusion: These data identified LY in vitro as an activator of Aβ degrading genes leading to cerebral Aβ load amelioration in early and late AD pathology. Although Aβ plaque reduction by LY failed in vivo, LY may be used as therapeutic agent to treat AD-related neuroinflammation and impaired neuronal integrity.

scholarly journals Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation

2004 ◽  
Vol 377 (3) ◽  
pp. 709-716 ◽  
Author(s):  
Emma T. A. S. JAIKARAN ◽  
Melanie R. NILSSON ◽  
Anne CLARK
Keyword(s):  
Type 2 Diabetes ◽  
Amyloid Fibrils ◽  
Secretory Granules ◽  
Pancreatic Beta Cell ◽  
Islet Amyloid Polypeptide ◽  
Fibril Formation ◽  
Islet Amyloid ◽  
Β Sheet

Islet amyloid polypeptide (IAPP), or ‘amylin’, is co-stored with insulin in secretory granules of pancreatic islet β-cells. In Type 2 diabetes, IAPP converts into a β-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop β-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation.

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