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1.
J Biol Chem ; 289(20): 13838-50, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24671416

RESUMO

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/farmacologia , Heparina/farmacologia , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/química , alfa-Sinucleína/toxicidade , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Estrutura Secundária de Proteína
2.
J Biol Chem ; 287(4): 2398-409, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22134915

RESUMO

Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of α-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote α-synuclein aggregation. Taking into account the toxicity of α-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.


Assuntos
Amiloide/química , Gliceraldeído-3-Fosfato Desidrogenases/química , Heparina/química , Multimerização Proteica , alfa-Sinucleína/química , Amiloide/metabolismo , Animais , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Doença de Parkinson/metabolismo , Coelhos , alfa-Sinucleína/metabolismo
3.
Sci Rep ; 7: 41755, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-28155912

RESUMO

Synucleinophaties are progressive neurodegenerative disorders with no cure to date. An attractive strategy to tackle this problem is repurposing already tested safe drugs against novel targets. In this way, doxycycline prevents neurodegeneration in Parkinson models by modulating neuroinflammation. However, anti-inflammatory therapy per se is insufficient to account for neuroprotection. Herein we characterise novel targets of doxycycline describing the structural background supporting its effectiveness as a neuroprotector at subantibiotic doses. Our results show that doxycycline reshapes α-synuclein oligomers into off-pathway, high-molecular-weight species that do not evolve into fibrils. Off-pathway species present less hydrophobic surface than on-pathway oligomers and display different ß-sheet structural arrangement. These structural changes affect the α-synuclein ability to destabilize biological membranes, cell viability, and formation of additional toxic species. Altogether, these mechanisms could act synergically giving novel targets for repurposing this drug.


Assuntos
Doxiciclina/farmacologia , Reposicionamento de Medicamentos , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Doxiciclina/uso terapêutico , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas , Ligação Proteica , Conformação Proteica em Folha beta , Multimerização Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , alfa-Sinucleína/química
4.
Curr Protein Pept Sci ; 12(3): 166-80, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21348838

RESUMO

Even though our knowledge of how proteins misfold and aggregate is deeper nowadays, the mechanisms driving this process are still poorly understood. Among the factors involved, membranes should be taken into account. Indeed, convincing evidence suggests that membranes may influence protein folding, misfolding and aggregation. In fact, membrane lipid composition of different cellular types may attenuate or intensify the environmental pressure over protein folding equilibrium. In the present review the aim is to make an up-to-date analysis of the membrane influence on protein aggregation from a biophysical point of view in order to provide useful tools for researchers from other fields. In particular, we discuss how membranes can alter protein environment, e.g. increasing local protein concentration, lowering pH and dielectric constant, allowing accessibility to the hydrophobic milieu and promoting surface crowding, all of which will lead to protein aggregation. In addition, we review the role that specific lipids may exert on protein aggregation and finally we analyse the possible implication of membrane-related oxidative stress on amyloidogenesis.


Assuntos
Amiloide/metabolismo , Membrana Celular/fisiologia , Modelos Biológicos , Biofísica , Membrana Celular/patologia , Humanos , Estresse Oxidativo , Deficiências na Proteostase/fisiopatologia , Termodinâmica
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