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1.
Photochem Photobiol ; 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38314442

RESUMO

In this study, we investigate the intricate regulatory mechanisms underlying the circadian clock in Drosophila, focusing on the light-induced conformational changes in the cryptochrome (DmCry). Upon light exposure, DmCry undergoes conformational changes that prompt its binding to Timeless and Jetlag proteins, initiating a cascade crucial for the starting of a new circadian cycle. DmCry is subsequently degraded, contributing to the desensitization of the resetting mechanism. The transient and short-lived nature of DmCry protein-protein interactions (PPIs), leading to DmCry degradation within an hour of light exposure, presents a challenge for comprehensive exploration. To address this, we employed proximity-dependent biotinylation techniques, combining engineered BioID (TurboID) and APEX (APEX2) enzymes with mass spectrometry. This approach enabled the identification of the in vitro DmCry interactome in Drosophila S2 cells, uncovering several novel PPIs associated with DmCry. Validation of these interactions through a novel co-immunoprecipitation technique enhances the reliability of our findings. Importantly, our study suggests the potential of this method to reveal additional circadian clock- or magnetic field-dependent PPIs involving DmCry. This exploration of the DmCry interactome not only advances our understanding of circadian clock regulation but also establishes a versatile framework for future investigations into light- and time-dependent protein interactions in Drosophila.

2.
Proteins ; 90(6): 1315-1330, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35122331

RESUMO

Circadian rhythms are a series of endogenous autonomous 24-h oscillations generated by the circadian clock. At the molecular level, the circadian clock is based on a transcription-translation feedback loop, in which BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME (CRY) and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. There are three PER proteins, of which PER2 shows the strongest oscillation at both stability and cellular localization level. Protein-protein interactions (PPIs) or interactome of the circadian clock proteins have been investigated using classical methods such as two-dimensional gel electrophoresis, immunoprecipitation-coupled mass spectrometry, and yeast-two hybrid assay where the dynamic and weak interactions are difficult to catch. To identify the interactome of PER2 we have adopted proximity-dependent labeling with biotin and mass spectrometry-based identification of labeled proteins (BioID). In addition to known interactions with such as CRY1 and CRY2, we have identified several new PPIs for PER2 and confirmed some of them using co-immunoprecipitation technique. This study characterizes the PER2 protein interactions in depth, and it also implies that using a fast BioID method with miniTurbo or TurboID coupled to other major circadian clock proteins might uncover other interactors in the clock that have yet to be discovered.


Assuntos
Relógios Circadianos , Proteínas Circadianas Period , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Proteoma/metabolismo
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