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1.
Nanoscale ; 11(18): 9185-9193, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31038146

RESUMO

Recent advances in nanotechnology have developed a lot of opportunities for biological applications. In this work, multifunctional MoS2/AuNR nanocomposites with unique high NIR absorption were designed via combining MoS2 nanosheets and gold nanorods (AuNRs). The nanocomposites were synthesized through electrostatic self-assembly and showed high stability and good biocompatibility. Then they were used to modulate the aggregation of amyloid-ß peptides, destabilize mature fibrils under NIR irradiation, and eliminate Aß-induced ROS against neurotoxicity. The inhibition and destabilization effects were confirmed by Thioflavin T (ThT) fluorescence assay and transmission electron microscopy (TEM). Cell viability assay and ROS assay revealed that MoS2/AuNR nanocomposites could alleviate Aß-induced oxidative stress and cell toxicity. More importantly, both MoS2 nanosheets and AuNRs can be used as NIR photothermal agents, MoS2/AuNR nanocomposites have enhanced ability of disrupting Aß fibrils and improved cell viability by generating local heat under low power NIR irradiation. Our results provide new insights into the design of new multifunctional systems for the treatment of amyloid-related diseases.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Raios Infravermelhos , Nanocompostos/química , Fragmentos de Peptídeos/metabolismo , Amiloide/química , Amiloide/toxicidade , Peptídeos beta-Amiloides/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dissulfetos/química , Ouro/química , Humanos , Molibdênio/química , Nanocompostos/toxicidade , Nanotubos/química , Fragmentos de Peptídeos/química , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
2.
Nat Commun ; 10(1): 62, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622276

RESUMO

The extension of the amyloid hypothesis to include non-protein metabolite assemblies invokes a paradigm for the pathology of inborn error of metabolism disorders. However, a direct demonstration of the assembly of metabolite amyloid-like structures has so far been provided only in vitro. Here, we established an in vivo model of adenine self-assembly in yeast, in which toxicity is associated with intracellular accumulation of the metabolite. Using a strain blocked in the enzymatic pathway downstream to adenine, we observed a non-linear dose-dependent growth inhibition. Both the staining with an indicative amyloid dye and anti-adenine assemblies antibodies demonstrated the accumulation of adenine amyloid-like structures, which were eliminated by lowering the supplied adenine levels. Treatment with a polyphenol inhibitor reduced the occurrence of amyloid-like structures while not affecting the dramatic increase in intracellular adenine concentration, resulting in inhibition of cytotoxicity, further supporting the notion that toxicity is triggered by adenine assemblies.


Assuntos
Adenina/metabolismo , Amiloide/metabolismo , Erros Inatos do Metabolismo/etiologia , Saccharomyces cerevisiae/metabolismo , Adenina/toxicidade , Amiloide/toxicidade , Erros Inatos do Metabolismo/metabolismo
3.
Int J Biol Macromol ; 118(Pt A): 552-560, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29949746

RESUMO

In the present study, we have investigated the effects of protein concentration and stirring on the in vitro assembly of Hen Egg White Lysozyme (HEWL), particularly with regard to the aggregate morphology and anti-amyloidogenic properties of two naturally occurring polyphenols, taxifolin and silibinin. The results obtained clearly demonstrated that applying stirring and concentration enhancement alter the amount as well as morphology of amyloid fibrils formed. Additionally, latter aggregates exhibited higher affinity for amyloid-specific dyes. The second part of the present investigation was devoted to studies involving anti-amyloidogenic properties of selected polyphenols. Importantly, we found that the potency of polyphenols to inhibit HEWL amyloid fibrillation and related toxicity is strongly dependent on the amyloidogenic conditions in which amyloid fibrils are produced. Based on obtained data, under condition where the rate of protein assembly is high (higher protein concentration and stirring), the capacity of polyphenols to inhibit HEWL fibrillogenesis and related cytotoxicity may dramatically decrease. Similar results were obtained when we used taxifolin to inhibit bovine insulin amyloid fibrillation. Additionally, amyloidogenic conditions may also affect the mechanism by which these molecules inhibit HEWL fibrillation. The possible mechanism by which selected polyphenols exert their inhibitory effects, under various experimental conditions, is also discussed.


Assuntos
Amiloide/química , Muramidase/química , Polifenóis/farmacologia , Agregados Proteicos/efeitos dos fármacos , Amiloide/toxicidade , Animais , Bovinos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Muramidase/toxicidade , Propriedades de Superfície
4.
Int J Biol Macromol ; 114: 830-835, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29626601

RESUMO

Mounting evidence indicates soluble Aß42 oligomers as the most toxic species causing neuronal death which leads to the onset and progression of Alzheimer disease (AD). Recently, it has been found that neurotoxic Aß42 oligomers grow from monomeric species or arise following secondary nucleation by preformed mature fibrils. Thus, the use of natural compounds such as polyphenols to hinder the growth or to remodel Aß42 fibrils is one of the most promising strategies for AD treatment. In our previous study, we showed that 1, 2, 4-trihydroxynaphthalene-2-O-ß-d-glucopyranoside (THNG) inhibits Aß42 aggregation during the early steps of the aggregation process, inhibits its conformational change to a ß-sheet-rich structure, decreases its polymerization, inhibits its fibrillogenisis and reduces oxidative stress and aggregate cytotoxicity. Here, we used different spectroscopic and cell culture methods to check the effect of THNG on fibrils disaggregation. We showed that THNG binds to mature Aß42 fibrils, rearrange their secondary structure, and remodels them into non-amyloid, less toxic, species by inhibiting their interaction with the plasma membrane. Our findings reveal that THNG is a good agent to remodel amyloid fibrils and could be used as a starting molecular scaffold to design new anti-AD drugs.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/efeitos dos fármacos , Lawsonia (Planta)/química , Fragmentos de Peptídeos/química , Amiloide/toxicidade , Peptídeos beta-Amiloides/toxicidade , Linhagem Celular Tumoral , Dicroísmo Circular , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Gangliosídeo G(M1)/análise , Humanos , Microdomínios da Membrana , Microscopia Eletrônica , Estrutura Molecular , Neuroblastoma/patologia , Fragmentos de Peptídeos/toxicidade , Folhas de Planta/química
5.
Rev Neurosci ; 29(6): 613-627, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-29447116

RESUMO

A major feature of Alzheimer's disease (AD) pathology is the plaque composed of aggregated amyloid-ß (Aß) peptide. Although these plaques may have harmful properties, there is much evidence to implicate soluble oligomeric Aß as the primary noxious form. Aß oligomers can be generated both extracellularly and intracellularly. Aß is toxic to neurons in a myriad of ways. It can cause pore formation resulting in the leakage of ions, disruption of cellular calcium balance, and loss of membrane potential. It can promote apoptosis, cause synaptic loss, and disrupt the cytoskeleton. Current treatments for AD are limited and palliative. Much research and effort is being devoted to reducing Aß production as an approach to slowing or preventing the development of AD. Aß formation results from the amyloidogenic cleavage of human amyloid precursor protein (APP). Reconfiguring this process to disfavor amyloid generation might be possible through the reduction of APP or inhibition of enzymes that convert the precursor protein to amyloid.


Assuntos
Doença de Alzheimer , Amiloide/metabolismo , Amiloide/toxicidade , Encéfalo/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Humanos
6.
Biochemistry ; 57(5): 791-804, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29286644

RESUMO

Aggregation of α-synuclein (α-Syn) into neurotoxic oligomers and amyloid fibrils is suggested to be the pathogenic mechanism for Parkinson's disease (PD). Recent studies have indicated that oligomeric species of α-Syn are more cytotoxic than their mature fibrillar counterparts, which are responsible for dopaminergic neuronal cell death in PD. Therefore, the effective therapeutic strategies for tackling aggregation-associated diseases would be either to prevent aggregation or to modulate the aggregation process to minimize the formation of toxic oligomers during aggregation. In this work, we showed that arginine-substituted α-Syn ligands, based on the most aggregation-prone sequence of α-Syn, accelerate the protein aggregation in a concentration-dependent manner. To elucidate the mechanism by which Arg-substituted peptides could modulate α-Syn aggregation kinetics, we performed surface plasmon resonance (SPR) spectroscopy, nuclear magnetic resonance (NMR) studies, and all-atom molecular dynamics (MD) simulation. The SPR analysis showed a high binding potency of these peptides with α-Syn but one that was nonspecific in nature. The two-dimensional NMR studies suggest that a large stretch within the C-terminus of α-Syn displays a chemical shift perturbation upon interacting with Arg-substituted peptides, indicating C-terminal residues of α-Syn might be responsible for this class of peptide binding. This is further supported by MD simulation studies in which the Arg-substituted peptide showed the strongest interaction with the C-terminus of α-Syn. Overall, our results suggest that the binding of Arg-substituted ligands to the highly acidic C-terminus of α-Syn leads to reduced charge density and flexibility, resulting in accelerated aggregation kinetics. This may be a potentially useful strategy while designing peptides, which act as α-Syn aggregation modulators.


Assuntos
Amiloide/química , Arginina/química , Fragmentos de Peptídeos/antagonistas & inibidores , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/prevenção & controle , alfa-Sinucleína/antagonistas & inibidores , Substituição de Aminoácidos , Amiloide/toxicidade , Linhagem Celular Tumoral , Desenho de Drogas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Neuroblastoma/patologia , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Domínios Proteicos , Ressonância de Plasmônio de Superfície , alfa-Sinucleína/química , alfa-Sinucleína/toxicidade
7.
J Am Chem Soc ; 139(47): 17098-17108, 2017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-29058422

RESUMO

The conversion of the native random coil amyloid beta (Aß) into amyloid fibers is thought to be a key event in the progression of Alzheimer's disease (AD). A significant body of evidence suggests that the highly dynamic Aß oligomers are the main causal agent associated with the onset of AD. Among many potential therapeutic approaches, one is the modulation of Aß conformation into off-pathway structures to avoid the formation of the putative neurotoxic Aß oligomers. A library of oligoquinolines was screened to identify antagonists of Aß oligomerization, amyloid formation, and cytotoxicity. A dianionic tetraquinoline, denoted as 5, was one of the most potent antagonists of Aß fibrillation. Biophysical assays including amyloid kinetics, dot blot, ELISA, and TEM show that 5 effectively inhibits both Aß oligomerization and fibrillation. The antagonist activity of 5 toward Aß aggregation diminishes with sequence and positional changes in the surface functionalities. 5 binds to the central discordant α-helical region and induces a unique α-helical conformation in Aß. Interestingly, 5 adjusts its conformation to optimize the antagonist activity against Aß. 5 effectively rescues neuroblastoma cells from Aß-mediated cytotoxicity and antagonizes fibrillation and cytotoxicity pathways of secondary nucleation induced by seeding. 5 is also equally effective in inhibiting preformed oligomer-mediated processes. Collectively, 5 induces strong secondary structure in Aß and inhibits its functions including oligomerization, fibrillation, and cytotoxicity.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Amiloide/química , Amiloide/toxicidade , Agregação Patológica de Proteínas/tratamento farmacológico , Doença de Alzheimer/metabolismo , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Humanos , Cinética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Príons/antagonistas & inibidores , Príons/química , Príons/metabolismo , Príons/toxicidade , Estrutura Secundária de Proteína/efeitos dos fármacos
8.
Cytotherapy ; 19(12): 1426-1437, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29037943

RESUMO

BACKGROUND AIMS: Light chain (AL) amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. Patients with LC amyloid involvement of the heart have the worst morbidity and mortality. Current treatments target the plasma cells to reduce further production of amyloid proteins. There is dire need to understand the mechanisms of cardiac tissue damage from amyloid to develop novel therapies. We recently reported that LC soluble and fibrillar species cause apoptosis and inhibit cell growth in human cardiomyocytes. Mesenchymal stromal cells (MSCs) can promote wound healing and tissue remodeling. The objective of this study was to evaluate MSCs to protect cardiomyocytes affected by AL amyloid fibrils. METHODS: We used live cell imaging and proteomics to analyze the effect of MSCs in the growth arrest caused by AL amyloid fibrils. RESULTS: We evaluated the growth of human cardiomyocytes (RFP-AC16 cells) in the presence of cytotoxic LC amyloid fibrils. MSCs reversed the cell growth arrest caused by LC fibrils. We also demonstrated that this effect requires cell contact and may be mediated through paracrine factors modulating cell adhesion and extracellular matrix remodeling. To our knowledge, this is the first report of MSC protection of human cardiomyocytes in amyloid disease. CONCLUSIONS: This important proof of concept study will inform future rational development of MSC therapy in cardiac LC amyloid.


Assuntos
Amiloide/toxicidade , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/patologia , Amiloide/metabolismo , Apoptose , Células Cultivadas , Técnicas de Cocultura , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/terapia , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo
9.
FASEB J ; 31(12): 5296-5306, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28821639

RESUMO

Human islet amyloid polypeptide (hIAPP) aggregation is associated with ß-cell dysfunction and death in type 2 diabetes (T2D). we aimed to determine whether in vivo treatment with chemical chaperone 4-phenylbutyrate (PBA) ameliorates hIAPP-induced ß-cell dysfunction and islet amyloid formation. Oral administration of PBA in hIAPP transgenic (hIAPP Tg) mice expressing hIAPP in pancreatic ß cells counteracted impaired glucose homeostasis and restored glucose-stimulated insulin secretion. Moreover, PBA treatment almost completely prevented the transcriptomic alterations observed in hIAPP Tg islets, including the induction of genes related to inflammation. PBA also increased ß-cell viability and improved insulin secretion in hIAPP Tg islets cultured under glucolipotoxic conditions. Strikingly, PBA not only prevented but even reversed islet amyloid deposition, pointing to a direct effect of PBA on hIAPP. This was supported by in silico calculations uncovering potential binding sites of PBA to monomeric, dimeric, and pentameric fibrillar structures, and by in vitro assays showing inhibition of hIAPP fibril formation by PBA. Collectively, these results uncover a novel beneficial effect of PBA on glucose homeostasis by restoring ß-cell function and preventing amyloid formation in mice expressing hIAPP in ß cells, highlighting the therapeutic potential of PBA for the treatment of T2D.-Montane, J., de Pablo, S., Castaño, C., Rodríguez-Comas, J., Cadavez, L., Obach, M., Visa, M., Alcarraz-Vizán, G., Sanchez-Martinez, M., Nonell-Canals, A., Parrizas, M., Servitja, J.-M., Novials, A. Amyloid-induced ß-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment.


Assuntos
Amiloide/toxicidade , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Fenilbutiratos/farmacologia , Animais , Teste de Tolerância a Glucose , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/ultraestrutura , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real
10.
Biochimie ; 142: 22-30, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28778718

RESUMO

Type 2 diabetes mellitus is characterized histopathologically by the presence of fibrillary amyloid deposits in the pancreatic islets of Langerhans. Human islet amyloid polypeptide (hIAPP), the 37-residue pancreatic hormone, is the major constituent of these amyloid deposits. The propensity of IAPP to form amyloid fibrils is strongly dependent on its primary sequence. An intriguing example is His at residue 18. Although H18 is located outside the amyloidogenic region, it has been suggested that this residue and its charge state play an important role in the kinetics of conformational changes and fibril formation as well as in mediating cell toxicity. To gain more insight into the importance of this residue, we have synthesized four analogues (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and we performed a full biophysical study on the properties of these peptides. Kinetic experiments as monitored by thioflavin-T fluorescence, transmission electron microscopy, circular dichroism and cell toxicity assays revealed that all variants are less fibrillogenic and less toxic than native hIAPP both at neutral pH and at low pH. This demonstrates that the effect of H18 in native IAPP is not simply determined by its charge state, but rather that residue 18 is important for specific intra- and intermolecular interactions that occur during fibril formation and that may involve charge, size and hydrophobicity. Furthermore, our results indicate that H18R-IAPP has a strong inhibiting effect on native hIAPP fibril formation. Together these results highlight the large impact of modifying a single residue outside the amyloidogenic domain on fibril formation and cell toxicity induced by IAPP, opening up new avenues for design of inhibitors or modulators of IAPP aggregation.


Assuntos
Amiloide/química , Amiloide/toxicidade , Ilhotas Pancreáticas/metabolismo , Agregados Proteicos , Sequência de Aminoácidos , Amiloide/genética , Animais , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Mutação , Ratos
11.
Chemistry ; 23(40): 9618-9624, 2017 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-28544138

RESUMO

Inhibiting the toxic aggregation of amyloid-ß and the tau protein, the key pathological agents involved in Alzheimer's, is a leading approach in modulating disease progression. Using an aggregative tau-derived model peptide, Ac-PHF6-NH2 , the substitution of its amino acids with proline, a known efficient ß-breaker, is shown to reduce self-assembly. This effect is attributed to the steric hindrance created by the proline substitution, which results in disruption of the ß-sheet formation process. Moreover, several of the proline-substituted peptides inhibit the aggregation of Ac-PHF6-NH2 amyloidogenic peptide. Two of these modified inhibitors also disassemble pre-formed Ac-PHF6-NH2 fibrils and one inhibits induced cytotoxicity of the fibrils. Taken together, these lead ß-breaker peptides may be developed into novel Alzheimer's disease therapeutics.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Prolina/química , Proteínas tau/química , Amiloide/metabolismo , Amiloide/toxicidade , Animais , Sobrevivência Celular , Humanos , Oligopeptídeos/metabolismo , Células PC12 , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica , Ratos , Proteínas tau/metabolismo
12.
Nat Commun ; 8: 15462, 2017 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-28537272

RESUMO

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells.


Assuntos
Amiloide/química , Proteína Huntingtina/genética , Doença de Huntington/genética , Peptídeos/química , Agregação Patológica de Proteínas/genética , Amiloide/genética , Amiloide/metabolismo , Amiloide/toxicidade , Animais , Linhagem Celular , Éxons/genética , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Proteína Huntingtina/toxicidade , Doença de Huntington/patologia , Espectroscopia de Ressonância Magnética , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Neurônios , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/toxicidade , Agregação Patológica de Proteínas/patologia , Estrutura Secundária de Proteína/genética
13.
Chem Asian J ; 12(10): 1062-1068, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28303660

RESUMO

A 39-amino acid peptide fragment that is derived from prostatic acidic phosphatase (PAP), PAP248-286 , is secreted in large amounts in human semen and forms amyloid fibrils. These fibrils can capture HIV virions and increase the attachment of virions to target cells; as such, they are called a "semen-derived enhancer of virus infection" (SEVI). Therefore, the inhibition of the formation of PAP248-286 amyloid fibrils is of great significance. Herein, we demonstrate that brazilin effectively inhibits PAP248-286 aggregation. The inhibitory effect increases with increasing brazilin concentration. Thioflavin T fluorescence assays and TEM observations confirmed that a few fibrils formed when brazilin was present with PAP248-286 in an equimolar concentration. Circular dichroism spectroscopy indicated that brazilin inhibited the secondary structural transitions from α-helices and random coils into ß-sheets. Cytotoxicity assays showed that brazilin significantly decreased the cytotoxicity of the fibrils at 0.01 mmol L-1 . Isothermal titration calorimetry revealed that hydrophobic interactions were the main driving force for the binding of brazilin to the PAP248-286 monomer (dissociation constant, 4.03 µmol L-1 ), and that the binding affinity of brazilin for the fibrils was at least three orders of magnitude lower than that for the monomer. These results indicate that brazilin holds great potential as a small-molecule agent against SEVIs.


Assuntos
Fosfatase Ácida/antagonistas & inibidores , Amiloide/antagonistas & inibidores , Amiloide/toxicidade , Benzopiranos/farmacologia , Fosfatase Ácida/metabolismo , Fosfatase Ácida/toxicidade , Amiloide/metabolismo , Animais , Benzopiranos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Células PC12 , Agregados Proteicos/efeitos dos fármacos , Ratos , Relação Estrutura-Atividade
14.
Nat Struct Mol Biol ; 24(4): 379-386, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28218748

RESUMO

Deposition of amyloid-ß plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-ß peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aß fibrils increases ß-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aß aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-ß-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Amiloide/toxicidade , Neurotoxinas/toxicidade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Amiloide/química , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Dicroísmo Circular , Imunofluorescência , Humanos , Camundongos Transgênicos , Microscopia de Força Atômica , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/química , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Ratos Sprague-Dawley , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
15.
Elife ; 62017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28045370

RESUMO

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic ß-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of ß-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile ß-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP.


Assuntos
Amiloide/química , Amiloide/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Microscopia Crioeletrônica , Humanos , Conformação Proteica em Folha beta
16.
Sci Rep ; 7: 40859, 2017 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28102321

RESUMO

The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington's disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson's disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism.


Assuntos
Amiloide/metabolismo , Chaperonina com TCP-1/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/toxicidade , Linhagem Celular Tumoral , Chaperonina com TCP-1/química , Humanos , Microscopia Eletrônica de Transmissão , Mutagênese Sítio-Dirigida , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética
17.
Phys Chem Chem Phys ; 19(2): 1458-1465, 2017 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-27982149

RESUMO

Amyloid ß is one of the peptides involved in the onset of Alzheimer's disease, yet the structure of the toxic species and its underlying mechanism remain elusive on account of the dynamic nature of the Aß oligomerisation process. While it has been reported that incubation of Amyloid ß (1-42) sequences (Aß42) lead to formation of aggregates that vary in morphology and toxicity, we demonstrate that addition of a discrete macrocyclic host molecule, cucurbit[8]uril (CB[8]), substantially reduces toxicity in the neuronal cell line SH-SY5Y. The macrocycle preferentially targets Phe residues in Aß42 complexing them in a 2 : 1 fashion in neighboring peptide strands. A small but significant structural 'switch' occurs, which induces an increased aggregation rate, suggesting a different cell-uptake mechanism for Aß42 in the presence of CB[8]. Dramatically increasing the rate of Aß42 aggregation with CB[8] bypasses the toxic, oligomeric state offering an alternative approach to counter Alzheimer's disease.


Assuntos
Amiloide/metabolismo , Amiloide/toxicidade , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Imidazóis/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Linhagem Celular , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Agregação Patológica de Proteínas
18.
Mol Cell Biochem ; 425(1-2): 85-93, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27804051

RESUMO

Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic ß-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 ß-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17-29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.


Assuntos
Amiloide/toxicidade , Cobre/toxicidade , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Proteínas do Tecido Nervoso/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Linhagem Celular , Humanos , Proteínas do Tecido Nervoso/genética , Ratos , Receptor para Produtos Finais de Glicação Avançada/genética , Receptores de Fator de Crescimento Neural/genética
19.
Nature ; 539(7628): 227-235, 2016 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-27830791

RESUMO

The aggregation of proteins into structures known as amyloids is observed in many neurodegenerative diseases, including Alzheimer's disease. Amyloids are composed of pairs of tightly interacting, many stranded and repetitive intermolecular ß-sheets, which form the cross-ß-sheet structure. This structure enables amyloids to grow by recruitment of the same protein and its repetition can transform a weak biological activity into a potent one through cooperativity and avidity. Amyloids therefore have the potential to self-replicate and can adapt to the environment, yielding cell-to-cell transmissibility, prion infectivity and toxicity.


Assuntos
Amiloide/química , Amiloide/metabolismo , Amiloide/biossíntese , Amiloide/toxicidade , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Animais , Humanos , Modelos Moleculares , Príons/biossíntese , Príons/química , Príons/classificação , Príons/toxicidade , Estrutura Secundária de Proteína , alfa-Sinucleína/biossíntese , alfa-Sinucleína/química , alfa-Sinucleína/toxicidade
20.
ACS Appl Mater Interfaces ; 8(21): 13309-19, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-27152771

RESUMO

Inhibitory modulation toward de novo protein aggregation is likely to be a vital and promising therapeutic strategy for understanding the molecular etiology of amyloid related diseases such as Alzheimer's disease (AD). The building up of toxic oligomeric and fibrillar amyloid aggregates in the brain plays host to a downstream of events, causing damage to axons, dendrites, synapses, signaling, transmission, and finally cell death. Herein, we introduce a novel conjugated polymer (CP), hydroxyquinoline appended polyfluorene (PF-HQ), which has a typical "amyloid like" surface motif and exhibits inhibitory modulation effect on amyloid ß (Aß) aggregation. We delineate inhibitory effects of PF-HQ based on Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), circular dichroism (CD), and Fourier transform infrared (FTIR) studies. The amyloid-like PF-HQ forms nano coaggregates by templating with toxic amyloid intermediates and displays improved inhibitory impacts toward Aß fibrillation and diminishes amyloid cytotoxicity. We have developed a CP based modulation strategy for the first time, which demonstrates beneficiary amyloid-like surface motif to interact efficiently with the protein, the pendant side groups to trap the toxic amyloid intermediates as well as optical signal to acquire the mechanistic insight.


Assuntos
Amiloide/metabolismo , Fluorenos/química , Fluorenos/farmacologia , Hidroxiquinolinas/química , Doença de Alzheimer , Motivos de Aminoácidos , Amiloide/química , Amiloide/toxicidade , Fragmentos de Peptídeos , Agregação Patológica de Proteínas/metabolismo , Ligação Proteica/efeitos dos fármacos
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