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1.
FEBS Lett ; 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31721178

RESUMO

The constituent paired helical filaments (PHFs) in neurofibrillary tangles are insoluble intracellular deposits central to the development of Alzheimer's disease (AD) and other tauopathies. Full-length tau requires the addition of anionic cofactors such as heparin to enhance assembly. We have shown that a fragment from the proteolytically stable core of the PHF, tau 297-391 known as 'dGAE', spontaneously forms cross-ß-containing PHFs and straight filaments under physiological conditions. Here, we have analysed and compared the structures of the filaments formed by dGAE in vitro with those deposited in the brains of individuals diagnosed with AD. We show that dGAE forms PHFs that share a macromolecular structure similar to those found in brain tissue. Thus, dGAEs may serve as a model system for studying core domain assembly and for screening for inhibitors of tau aggregation.

2.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140257, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31352057

RESUMO

The formation of a diverse range of amyloid structures from normally soluble proteins and peptides is a hallmark of devastating human disorders as well as biological functions. The current molecular understanding of the amyloid lifecycle reveals four processes central to their growth and propagation: primary nucleation, elongation, secondary nucleation and division. However, these processes result in a wide range of cross-ß packing and filament arrangements, including diverse assemblies formed from identical monomeric precursors with the same amino acid sequences. Here, we review current structural and mechanistic understanding of amyloid self-assembly, and discuss how mesoscopic, i.e. micrometre to nanometre, organisation of amyloid give rise to suprastructural features that may be the key link between the polymorphic amyloid structures and the biological response they elicit. A greater understanding of the mechanisms governing suprastructure formation will guide future strategies to combat amyloid associated disorders and to use and control the amyloid quaternary structure in synthetic biology and materials applications.


Assuntos
Amiloide , Amiloidose , Polimorfismo Genético , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Amiloidose/genética , Amiloidose/metabolismo , Humanos , Estrutura Quaternária de Proteína
3.
J Mol Biol ; 431(12): 2248-2265, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31051176

RESUMO

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-ß amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset.

4.
Int J Parasitol ; 48(3-4): 197-201, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29195082

RESUMO

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.


Assuntos
Cryptosporidium parvum/crescimento & desenvolvimento , Animais , Linhagem Celular Tumoral , Células Cultivadas/parasitologia , Criopreservação , Cryptosporidium parvum/classificação , Humanos , Lipídeos/fisiologia , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Oocistos/classificação , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo
5.
ACS Nano ; 12(2): 1420-1432, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29275624

RESUMO

Understanding how molecules in self-assembled soft-matter nanostructures are organized is essential for improving the design of next-generation nanomaterials. Imaging these assemblies can be challenging and usually requires processing, e.g., staining or embedding, which can damage or obscure features. An alternative is to use bioinspired mineralization, mimicking how certain organisms use biomolecules to template mineral formation. Previously, we have reported the design and characterization of Self-Assembled peptide caGEs (SAGEs) formed from de novo peptide building blocks. In SAGEs, two complementary, 3-fold symmetric, peptide hubs combine to form a hexagonal lattice, which curves and closes to form SAGE nanoparticles. As hexagons alone cannot tile onto spheres, the network must also incorporate nonhexagonal shapes. While the hexagonal ultrastructure of the SAGEs has been imaged, these defects have not been observed. Here, we show that positively charged SAGEs biotemplate a thin, protective silica coating. Electron microscopy shows that these SiO2-SAGEs do not collapse, but maintain their 3D shape when dried. Atomic force microscopy reveals a network of hexagonal and irregular features on the SiO2-SAGE surface. The dimensions of these (7.2 nm ± 1.4 nm across, internal angles 119.8° ± 26.1°) are in accord with the designed SAGE network and with coarse-grained modeling of the SAGE assembly. The SiO2-SAGEs are permeable to small molecules (<2 nm), but not to larger biomolecules (>6 nm). Thus, bioinspired silicification offers a mild technique that preserves soft-matter nanoparticles for imaging, revealing structural details <10 nm in size, while also maintaining desirable properties, such as permeability to small molecules.


Assuntos
Peptídeos/síntese química , Dióxido de Silício/química , Tamanho da Partícula , Peptídeos/química , Propriedades de Superfície
6.
Nat Chem Biol ; 14(2): 142-147, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29227472

RESUMO

We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Álcool Desidrogenase/metabolismo , Bacillus/metabolismo , Proteínas de Bactérias/genética , Citoplasma/metabolismo , Escherichia coli/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Domínios Proteicos , Piruvato Descarboxilase/metabolismo
7.
Elife ; 62017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28880146

RESUMO

Transmissible amyloid particles called prions are associated with infectious prion diseases in mammals and inherited phenotypes in yeast. All amyloid aggregates can give rise to potentially infectious seeds that accelerate their growth. Why some amyloid seeds are highly infectious prion particles while others are less infectious or even inert, is currently not understood. To address this question, we analyzed the suprastructure and dimensions of synthetic amyloid fibrils assembled from the yeast (Saccharomyces cerevisiae) prion protein Sup35NM. We then quantified the ability of these particles to induce the [PSI+] prion phenotype in cells. Our results show a striking relationship between the length distribution of the amyloid fibrils and their ability to induce the heritable [PSI+] prion phenotype. Using a simple particle size threshold model to describe transfection activity, we explain how dimensions of amyloid fibrils are able to modulate their infectious potential as prions.


Assuntos
Amiloide/química , Fatores de Terminação de Peptídeos/química , Príons/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Amiloide/metabolismo , Amiloidose , Fatores de Terminação de Peptídeos/metabolismo , Fenótipo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Sci Rep ; 6: 39171, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27982082

RESUMO

Parkinson's disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable α-synuclein (α-syn) amyloid fibrils. α-synuclein is an intrinsically disordered protein that has the capacity to assemble to form ß-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients. In vitro, we show that dityrosine cross-links in α-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to the stabilization of α-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of α-syn in PD.


Assuntos
Corpos de Lewy/metabolismo , Doença de Parkinson/patologia , Tirosina/análogos & derivados , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Encéfalo/metabolismo , Cobre/química , Dimerização , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Oxirredução , Estresse Oxidativo , Doença de Parkinson/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em Tandem , Tirosina/análise , Tirosina/química , alfa-Sinucleína/química , alfa-Sinucleína/genética
9.
Sci Rep ; 6: 36899, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27845382

RESUMO

Bacterial microcompartments (BMCs) are proteinaceous organelles that are found in a broad range of bacteria and are composed of an outer shell that encases an enzyme cargo representing a specific metabolic process. The outer shell is made from a number of different proteins that form hexameric and pentameric tiles, which interact to allow the formation of a polyhedral edifice. We have previously shown that the Citrobacter freundii BMC associated with 1,2-propanediol utilization can be transferred into Escherichia coli to generate a recombinant BMC and that empty BMCs can be formed from just the shell proteins alone. Herein, a detailed structural and proteomic characterization of the wild type BMC is compared to the recombinant BMC and a number of empty BMC variants by 2D-gel electrophoresis, mass spectrometry, transmission electron microscopy (TEM) and atomic force microscopy (AFM). Specifically, it is shown that the wild type BMC and the recombinant BMC are similar in terms of composition, size, shape and mechanical properties, whereas the empty BMC variants are shown to be smaller, hollow and less malleable.


Assuntos
Citrobacter freundii/metabolismo , Organelas/química , Proteínas de Bactérias/metabolismo , Bioengenharia , Citrobacter freundii/química , Citrobacter freundii/ultraestrutura , Organelas/metabolismo , Organelas/ultraestrutura , Propilenoglicol/metabolismo , Proteômica
10.
J Chem Phys ; 144(17): 175101, 2016 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-27155653

RESUMO

Self-assembly of proteins into amyloid aggregates is an important biological phenomenon associated with human diseases such as Alzheimer's disease. Amyloid fibrils also have potential applications in nano-engineering of biomaterials. The kinetics of amyloid assembly show an exponential growth phase preceded by a lag phase, variable in duration as seen in bulk experiments and experiments that mimic the small volumes of cells. Here, to investigate the origins and the properties of the observed variability in the lag phase of amyloid assembly currently not accounted for by deterministic nucleation dependent mechanisms, we formulate a new stochastic minimal model that is capable of describing the characteristics of amyloid growth curves despite its simplicity. We then solve the stochastic differential equations of our model and give mathematical proof of a central limit theorem for the sample growth trajectories of the nucleated aggregation process. These results give an asymptotic description for our simple model, from which closed form analytical results capable of describing and predicting the variability of nucleated amyloid assembly were derived. We also demonstrate the application of our results to inform experiments in a conceptually friendly and clear fashion. Our model offers a new perspective and paves the way for a new and efficient approach on extracting vital information regarding the key initial events of amyloid formation.


Assuntos
Amiloide/química , Modelos Químicos , Humanos , Polimerização
11.
Biophys J ; 109(10): 1999-2000, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26588558
12.
PLoS One ; 10(7): e0132309, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26172440

RESUMO

There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity.


Assuntos
Amiloide/metabolismo , Amiloide/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fenômenos Ópticos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Linhagem Celular Tumoral , Humanos , Lipossomos/metabolismo , Muramidase/metabolismo , Muramidase/toxicidade , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Ligação Proteica , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade
13.
J Biol Chem ; 289(52): 35781-94, 2014 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-25378395

RESUMO

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of ß2-microglobulin (ß2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented ß2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented ß2m fibrils did not, however, cause cell death. Instead, fragmented ß2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.


Assuntos
Amiloide/fisiologia , Lisossomos/metabolismo , Proteólise , Microglobulina beta-2/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Nanopartículas/metabolismo , Oxirredução , Permeabilidade , Transporte Proteico
14.
PLoS One ; 9(8): e104492, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25100247

RESUMO

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ß2-microglobulin (ß2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ß2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ß2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ß2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ß2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ß2m amyloid-associated osteoarticular tissue destruction in DRA.


Assuntos
Amiloide/química , Endossomos/química , Membranas Intracelulares/química , Microglobulina beta-2/química , Amiloide/genética , Amiloide/metabolismo , Amiloidose/etiologia , Amiloidose/genética , Amiloidose/metabolismo , Endossomos/genética , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Membranas Artificiais , Diálise Renal/efeitos adversos , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
15.
ACS Synth Biol ; 3(7): 454-465, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24933391

RESUMO

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.


Assuntos
Reatores Biológicos , Etanol/metabolismo , Peptídeos/química , Aldeído Oxirredutases/química , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citrobacter freundii/enzimologia , Espectroscopia de Ressonância Magnética , Engenharia Metabólica , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Piruvato Descarboxilase/química , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
16.
Prion ; 8(2)2014.
Artigo em Inglês | MEDLINE | ID: mdl-24819071

RESUMO

Self-assembly of proteins and peptides into amyloid structures has been the subject of intense and focused research due to their association with neurodegenerative, age-related human diseases and transmissible prion diseases in humans and mammals. Of the disease associated amyloid assemblies, a diverse array of species, ranging from small oligomeric assembly intermediates to fibrillar structures, have been shown to have toxic potential. Equally, a range of species formed by the same disease associated amyloid sequences have been found to be relatively benign under comparable monomer equivalent concentrations and conditions. In recent years, an increasing number of functional amyloids have also been found. These developments show that not all amyloid structures are generically toxic to cells. Given these observations, it is important to understand why amyloid structures may encode such varied toxic potential despite sharing a common core molecular architecture. Here, we discuss possible links between different aspects of amyloidogenic structures and assembly mechanisms with their varied functional effects. We propose testable hypotheses for the relationship between amyloid structure and its toxic potential in the context of recent reports on amyloid sequence, structure, and toxicity relationships.


Assuntos
Amiloide/química , Amiloide/toxicidade , Humanos , Relação Estrutura-Atividade
17.
Chem Biol ; 21(2): 172-3, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24560165

RESUMO

Prion proteins can be propagated as amyloid fibrils with several different conformational variants. By providing structural information at atomic level for two such variants of a yeast prion, Frederick and colleagues, in this issue of Chemistry & Biology, reveal how conformational flexibility can generate phenotypic diversity.


Assuntos
Amiloide/metabolismo , Chaperonas Moleculares/metabolismo , Príons/metabolismo
18.
Biophys J ; 105(12): 2811-9, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24359753

RESUMO

Delineating the nanoscale properties and the dynamic assembly and disassembly behaviors of amyloid fibrils is key for technological applications that use the material properties of amyloid fibrils, as well as for developing treatments of amyloid-associated disease. However, quantitative mechanistic understanding of the complex processes involving these heterogeneous supramolecular systems presents challenges that have yet to be resolved. Here, we develop an approach that is capable of resolving the time dependence of fibril particle concentration, length distribution, and length and position dependence of fibril fragmentation rates using a generic mathematical framework combined with experimental data derived from atomic force microscopy analysis of fibril length distributions. By application to amyloid assembly of ß2-microglobulin in vitro under constant mechanical stirring, we present a full description of the fibril fragmentation and growth behavior, and demonstrate the predictive power of the approach in terms of the samples' fibril dimensions, fibril load, and their efficiency to seed the growth of new amyloid fibrils. The approach developed offers opportunities to determine, quantify, and predict the course and the consequences of amyloid assembly.


Assuntos
Amiloide/química , Microscopia de Força Atômica/métodos , Modelos Biológicos , Microglobulina beta-2/química
19.
Biophys J ; 105(3): 745-55, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23931322

RESUMO

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer's disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of ß2-microglobulin (ß2m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of ß2m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.


Assuntos
Membrana Celular/metabolismo , Polimerização/efeitos dos fármacos , Microglobulina beta-2/metabolismo , Catequina/análogos & derivados , Catequina/farmacologia , Heparina/farmacologia , Humanos , Polifenóis/farmacologia , Ligação Proteica/efeitos dos fármacos , Lipossomas Unilamelares/metabolismo , Microglobulina beta-2/química
20.
Proc Natl Acad Sci U S A ; 109(50): 20455-60, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23184970

RESUMO

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer's, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from ß(2)-microglobulin (ß(2)m). Using cryoelectron tomography, we demonstrate that fragmented ß(2)m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15-25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid-protein interactions in membrane remodelling.


Assuntos
Amiloide/química , Amiloide/ultraestrutura , Animais , Fenômenos Biofísicos , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Humanos , Lipossomos/química , Lipossomos/ultraestrutura , Membranas/química , Membranas/ultraestrutura , Microscopia de Fluorescência , Multimerização Proteica , Microglobulina beta-2/química , Microglobulina beta-2/ultraestrutura
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