Functionalization of amyloid fibrils via the Bri2 BRICHOS domain

AbstractAmyloid fibrils are mechanically robust and partly resistant to proteolytic degradation, making them potential candidates for scaffold materials in cell culture, tissue engineering, drug delivery and other applications. Such applications of amyloids would benefit from the possibility to functionalize the fibrils, for example by adding growth factors or cell attachment sites. The BRICHOS domain is found in a family of human proteins that harbor particularly amyloid-prone regions and can reduce aggregation as well as toxicity of several different amyloidogenic peptides. Recombinant human (rh) BRICHOS domains have been shown to bind to the surface of amyloid-β (Aβ) fibrils by immune electron microscopy. Here we produce fusion proteins between mCherry and rh Bri2 BRICHOS and show that they can bind to different amyloid fibrils with retained fluorescence of mCherry in vitro as well as in cultured cells. This suggests a “generic” ability of the BRICHOS domain to bind fibrillar surfaces that can be used to synthesize amyloid decorated with different protein functionalities.

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Redox-Dependent Copper Ion Modulation of Amyloid-β (1-42) Aggregation In Vitro

Biomolecules ◽

10.3390/biom10060924 ◽

2020 ◽

Vol 10 (6) ◽

pp. 924

Author(s):

Nima Sasanian ◽

David Bernson ◽

Istvan Horvath ◽

Pernilla Wittung-Stafshede ◽

Elin K. Esbjörner

Keyword(s):

Amyloid Fibrils ◽

Copper Ions ◽

Copper Ion ◽

Amyloid Β ◽

State Dependence ◽

Theoretical Approaches ◽

Aβ Aggregation ◽

Ion Effects ◽

Aβ Fibrils

Plaque deposits composed of amyloid-β (Aβ) fibrils are pathological hallmarks of Alzheimer’s disease (AD). Although copper ion dyshomeostasis is apparent in AD brains and copper ions are found co-deposited with Aβ peptides in patients’ plaques, the molecular effects of copper ion interactions and redox-state dependence on Aβ aggregation remain elusive. By combining biophysical and theoretical approaches, we here show that Cu2+ (oxidized) and Cu+ (reduced) ions have opposite effects on the assembly kinetics of recombinant Aβ(1-42) into amyloid fibrils in vitro. Cu2+ inhibits both the unseeded and seeded aggregation of Aβ(1-42) at pH 8.0. Using mathematical models to fit the kinetic data, we find that Cu2+ prevents fibril elongation. The Cu2+-mediated inhibition of Aβ aggregation shows the largest effect around pH 6.0 but is lost at pH 5.0, which corresponds to the pH in lysosomes. In contrast to Cu2+, Cu+ ion binding mildly catalyzes the Aβ(1-42) aggregation via a mechanism that accelerates primary nucleation, possibly via the formation of Cu+-bridged Aβ(1-42) dimers. Taken together, our study emphasizes redox-dependent copper ion effects on Aβ(1-42) aggregation and thereby provides further knowledge of putative copper-dependent mechanisms resulting in AD.

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Phenolic Acids Exert Anticholinesterase and Cognition-Improving Effects

Current Alzheimer Research ◽

10.2174/1567205014666171128102557 ◽

2018 ◽

Vol 15 (6) ◽

pp. 531-543 ◽

Cited By ~ 4

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.

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

Journal of Alzheimer s Disease Reports ◽

10.3233/adr-200247 ◽

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.

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

Biomolecules ◽

10.3390/biom11121848 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1848

Author(s):

Jacob Fritzsch ◽

Alexander Korn ◽

Dayana Surendran ◽

Martin Krueger ◽

Holger A. Scheidt ◽

...

Keyword(s):

Amyloid Fibrils ◽

Amyloid Β ◽

Neuronal Cell ◽

Cell Toxicity ◽

X Ray Diffraction ◽

Hydrophobic Contact ◽

Aβ Peptides ◽

Tht Fluorescence ◽

Modified Peptides ◽

Aβ Fibrils

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.

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

Molecules ◽

10.3390/molecules26206120 ◽

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.

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Alpha-synuclein from patient Lewy bodies exhibits distinct pathological activity that can be propagated in vitro

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01288-2 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Nicholas P. Marotta ◽

Jahan Ara ◽

Norihito Uemura ◽

Marshall G. Lougee ◽

Emily S. Meymand ◽

...

Keyword(s):

Cellular Response ◽

Amyloid Fibrils ◽

Cultured Cells ◽

Biological Activities ◽

Lewy Bodies ◽

Alpha Synuclein ◽

Biological Behavior ◽

Motor Deficits ◽

Original Material

AbstractLewy bodies (LBs) are complex, intracellular inclusions that are common pathological features of many neurodegenerative diseases. They consist largely of aggregated forms of the protein alpha-Synuclein (α-Syn), which misfolds to give rise to beta-sheet rich amyloid fibrils. The aggregation of monomers into fibrils occurs readily in vitro and pre-formed fibrils (PFFs) generated from recombinant α-Syn monomers are the basis of many models of LB diseases. These α-Syn PFFs recapitulate many pathological phenotypes in both cultured cells and animal models including the formation of α-Syn rich, insoluble aggregates, neuron loss, and motor deficits. However, it is not clear how closely α-Syn PFFs recapitulate the biological behavior of LB aggregates isolated directly from patients. Direct interrogation of the cellular response to LB-derived α-Syn has thus far been limited. Here we demonstrate that α-Syn aggregates derived from LB disease patients induce pathology characterized by a prevalence of large somatic inclusions that is distinct from the primarily neuritic pathology induced by α-Syn PFFs in our cultured neuron model. Moreover, these LB-derived aggregates can be amplified in vitro using recombinant α-Syn to generate aggregates that maintain the unique, somatic pathological phenotype of the original material. Amplified LB aggregates also showed greater uptake in cultured neurons and greater pathological burden and more rapid pathological spread in injected mouse brains, compared to α-Syn PFFs. Our work indicates that LB-derived α-Syn from diseased brains represents a distinct conformation species with unique biological activities that has not been previously observed in fully recombinant α-Syn aggregates and demonstrate a new strategy for improving upon α-Syn PFF models of synucleinopathies using amplified LBs.

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A cellular complex of BACE1 and γ-secretase sequentially generates Aβ from its full-length precursor

The Journal of Cell Biology ◽

10.1083/jcb.201806205 ◽

2019 ◽

Vol 218 (2) ◽

pp. 644-663 ◽

Cited By ~ 25

Author(s):

Lei Liu ◽

Li Ding ◽

Matteo Rovere ◽

Michael S. Wolfe ◽

Dennis J. Selkoe

Keyword(s):

Molecular Weight ◽

Cultured Cells ◽

Amyloid Β ◽

Full Length ◽

Aβ Peptides ◽

Whole Cells ◽

Sequential Processing ◽

Catalytically Active ◽

Cellular Complex

Intramembrane proteolysis of transmembrane substrates by the presenilin–γ-secretase complex is preceded and regulated by shedding of the substrate’s ectodomain by α- or β-secretase. We asked whether β- and γ-secretases interact to mediate efficient sequential processing of APP, generating the amyloid β (Aβ) peptides that initiate Alzheimer’s disease. We describe a hitherto unrecognized multiprotease complex containing active β- and γ-secretases. BACE1 coimmunoprecipitated and cofractionated with γ-secretase in cultured cells and in mouse and human brain. An endogenous high molecular weight (HMW) complex (∼5 MD) containing β- and γ-secretases and holo-APP was catalytically active in vitro and generated a full array of Aβ peptides, with physiological Aβ42/40 ratios. The isolated complex responded properly to γ-secretase modulators. Alzheimer’s-causing mutations in presenilin altered the Aβ42/40 peptide ratio generated by the HMW β/γ-secretase complex indistinguishably from that observed in whole cells. Thus, Aβ is generated from holo-APP by a BACE1–γ-secretase complex that provides sequential, efficient RIP processing of full-length substrates to final products.

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Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue

Nature Communications ◽

10.1038/s41467-019-12683-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 85

Author(s):

Marius Kollmer ◽

William Close ◽

Leonie Funk ◽

Jay Rasmussen ◽

Aref Bsoul ◽

...

Keyword(s):

Electron Microscopy ◽

Brain Tissue ◽

Amyloid Fibrils ◽

Structural Basis ◽

Amyloid Angiopathy ◽

Cryo Electron Microscopy ◽

Aβ Amyloid ◽

Cerebral Amyloid ◽

Aβ Fibrils

Abstract The formation of Aβ amyloid fibrils is a neuropathological hallmark of Alzheimer’s disease and cerebral amyloid angiopathy. However, the structure of Aβ amyloid fibrils from brain tissue is poorly understood. Here we report the purification of Aβ amyloid fibrils from meningeal Alzheimer’s brain tissue and their structural analysis with cryo-electron microscopy. We show that these fibrils are polymorphic but consist of similarly structured protofilaments. Brain derived Aβ amyloid fibrils are right-hand twisted and their peptide fold differs sharply from previously analyzed Aβ fibrils that were formed in vitro. These data underscore the importance to use patient-derived amyloid fibrils when investigating the structural basis of the disease.

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Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

Journal of Virology ◽

10.1128/jvi.75.11.5335-5342.2001 ◽

2001 ◽

Vol 75 (11) ◽

pp. 5335-5342 ◽

Cited By ~ 46

Author(s):

Kartik Chandran ◽

Xing Zhang ◽

Norman H. Olson ◽

Stephen B. Walker ◽

James D. Chappell ◽

...

Keyword(s):

Cultured Cells ◽

Cell Attachment ◽

Molecular Genetic ◽

Three Dimensional ◽

Host Cells ◽

Dimensional Structure ◽

Dependent Manner ◽

Viral Attachment ◽

Structure Assembly

ABSTRACT Mammalian reoviruses, prototype members of theReoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.

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Blood-Brain Barrier Permeable 2-Phenylbenzothiazolyl Iridi-um Complexes as Inhibitors and Probes of Aβ Aggregation

10.26434/chemrxiv.13606034 ◽

2021 ◽

Author(s):

Yiran Huang ◽

Hanah Na ◽

Liang Sun ◽

Karna Terpstra ◽

Kai Gui ◽

...

Keyword(s):

Blood Brain Barrier ◽

Day Treatment ◽

Amyloid Β ◽

Brain Barrier ◽

Blood Brain ◽

Aβ Aggregation ◽

D Values ◽

Aβ Fibrils

The aggregation of amyloid β (Aβ) peptides is a significant hallmark of Alzheimer’s Disease (AD) and the inhibition and detection of Aβ aggregates are important for the treatment and diagnosis of AD. Herein, a series of benzothiazole-based luminescent Ir(III) complexes HN-1 to HN-8 were reported, which exhibit appreciable Aβ aggregation inhibition ability in vitro and in living cells. In addition, they are capable of inducing a fluorescence turn-on effect when binding to Aβ fibrils and oligomers. Most importantly, compared to previously reported cationic metal complexes, the neutral Ir complexes reported here show optimal Log D values, which suggest these compounds should have enhanced blood brain barrier (BBB) permeability. Most importantly, in vivo studies show that the neutral Ir complexes HN-2, HN-3, and HN-8 successfully penetrate the BBB and stain amyloid plaques in AD mice brains after a 10-day treatment via i.p. injection, which is unprecedented for Ir(III) complexes, and thus can be used as lead compounds for AD therapeutics development.

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