RESUMO
Protein aggregation is the principal component of numerous protein misfolding pathologies termed proteinopathies, such as Alzheimer's disease, Parkinson's disease, prion disease, and AA amyloidosis with unmet treatment needs. Protein aggregation inhibitors have great potential for the prevention and treatment of proteinopathies. Here we report the development of an automated real-time microliter-scale high throughput screening (MSHTS) system for amyloid aggregation inhibitors using quantum-dot nanoprobes. Screening 504 crude extracts and 134 low molecular weight aromatic compounds revealed the relationship of amyloid-ß (Aß) aggregation inhibitory activities of plant extracts using a plant-based classification. Within the eudicots, rosids, Geraniales and Myrtales showed higher activity. Screening low molecular weight aromatic compounds demonstrated that the structure of tropolone endows it with potential Aß aggregation inhibitory activity. The activity of the most active tropolone derivative was higher than that of rosmarinic acid. MSHTS also identified three chaperone molecules as tau aggregation inhibitors. These results demonstrate that our automated MSHTS system is a novel and robust tool that can be adapted to a wide range of compounds and aggregation-prone polypeptides.
Assuntos
Neuropatias Amiloides/tratamento farmacológico , Proteínas Amiloidogênicas/antagonistas & inibidores , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Doenças Neurodegenerativas/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Agregação Patológica de Proteínas/tratamento farmacológico , Humanos , Pontos QuânticosRESUMO
The mammalian microtubule-associated proteins (MAPs), MAP2, MAP4, and τ, are structurally similar and considered to be evolutionarily related. The primary structure of a nematode MAP, PTL-1, also reportedly resembles those of the MAPs, but only in a small portion of the molecule. In this study, we elucidated the overall domain organization of PTL-1, using a molecular dissection technique. Firstly, we isolated nematode microtubules and proved that the recombinant PTL-1 binds to nematode and porcine microtubules with similar affinities. Then, the recombinant PTL-1 was genetically dissected to generate four shorter polypeptides, and their microtubule-binding and assembly promoting activities were assessed, using porcine microtubules and tubulin. PTL-1 was found to consist of two parts, microtubule-binding and projection domains, with the former further divided into three functionally distinct subdomains. The molecular architecture of PTL-1 was proved to be quite analogous to its mammalian counterparts, MAP2, MAP4, and τ, strongly supporting their evolutionary relationships.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas Associadas aos Microtúbulos/química , Microtúbulos/metabolismo , Tubulina (Proteína)/química , Proteínas tau/química , Animais , Sítios de Ligação , Evolução Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Suínos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
We previously used nuclear magnetic resonance (NMR) to analyze the structure of a synthetic tricosapeptide corresponding to an active site of microtubule-associated protein 4 (MAP4). To further the structural analysis, we have constructed a minimal active domain fragment of MAP4, encompassing the entire active site, and obtained its NMR spectra. The secondary structure prediction using partially assigned NMR data suggested that the fragment is largely unfolded. Two other independent techniques also demonstrated its unfolded nature, indicating that MAP4 belongs to the class of intrinsically disordered proteins (IDPs). The NMR spectra of the fragment-microtubule mixture revealed that the fragment binds to the microtubule using multiple binding sites, apparently contradicting our previous quantitative studies. Given that MAP4 is intrinsically disordered, we propose a mechanism in which any one of the binding sites is active at a time, which is one of the typical interaction mechanisms proposed for IDPs.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Fragmentos de Peptídeos/genética , Domínio Catalítico , Espectroscopia de Ressonância Magnética , Proteínas Associadas aos Microtúbulos/química , Microtúbulos/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
To analyze the interaction between microtubule-associated protein (MAP) 4 and microtubules physicochemically, a MAP4 active site fragment was designed for nuclear magnetic resonance (NMR) use. The fragment was bacterially expressed and purified to homogeneity. The buffer conditions for NMR were optimized to support microtubule assembly. The fragment was found to bind to microtubules under the optimized buffer conditions.
