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1.
Nat Commun ; 11(1): 605, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001718

RESUMO

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.


Assuntos
Optogenética , Proteínas/metabolismo , Actinas/metabolismo , Movimento Celular/efeitos da radiação , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Luz , Engenharia de Proteínas
2.
Int J Biol Macromol ; 125: 244-255, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30529354

RESUMO

pH-induced structural changes of the synthetic homopolypeptides poly-E, poly-K, poly-R, and intrinsically disordered proteins (IDPs) prothymosin α (ProTα) and linker histone H1, in concentrated PEG solutions simulating macromolecular crowding conditions within the membrane-less organelles, were characterized. The conformational transitions of the studied poly-amino acids in the concentrated PEG solutions depend on the polymerization degree of these homopolypeptides, the size of their side chains, the charge distribution of the side chains, and the crowding agent concentration. The results obtained for poly-amino acids are valid for IDPs having a significant total charge. The overcrowded conditions promote a significant increase in the cooperativity of the pH-induced coil-α-helix transition of ProTα and provoke histone H1 aggregation. The most favorable conditions for the pH-induced structural transitions in concentrated PEG solutions are realized when the charged residues are grouped in blocks, and when the distance between the end of the side group carrying charge and the backbone is small. Therefore, the block-wise distribution of charged residues within the IDPs not only plays an important role in the liquid-liquid phase transitions, but may also define the expressivity of structural transitions of these proteins in the overcrowded conditions of the membrane-less organelles.


Assuntos
Aminoácidos/química , Concentração de Íons de Hidrogênio , Proteínas Intrinsicamente Desordenadas/química , Peptídeos/química , Dobramento de Proteína , Amiloide/química , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Peptídeos/isolamento & purificação , Polietilenoglicóis/química , Conformação Proteica , Análise Espectral
3.
Chembiochem ; 19(12): 1334-1340, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29465801

RESUMO

Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner, QPAS1, is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested the functionality of two BphP1-QPAS1-based optogenetic tools-an NIR- and blue-light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA)-in several cell types, including cortical neurons. We found that the performance of these optogenetic tools often relied on physiological properties of a specific cell type, such as nuclear transport, which could limit the applicability of the blue-light-sensitive component of iRIS. In contrast, the NIR-light-sensing component of iRIS performed well in all tested cell types. The TA system showed the best performance in cervical cancer (HeLa), bone cancer (U-2 OS), and human embryonic kidney (HEK-293) cells. The small size of the QPAS1 component allowed the design of adeno-associated virus (AAV) particles, which were applied to deliver the TA system to neurons.


Assuntos
Neurônios/metabolismo , Optogenética/métodos , Proteínas/genética , Ativação Transcricional/efeitos da radiação , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Expressão Gênica/efeitos da radiação , Células HEK293 , Células HeLa , Humanos , Raios Infravermelhos , Luz , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Fitocromo/análise , Fitocromo/genética , Engenharia de Proteínas/métodos , Proteínas/análise , Ratos
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