How is the inner circadian clock controlled by interactive clock proteins?

Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type–dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

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Circadian Clock Proteins in Prokaryotes: Hidden Rhythms?

Frontiers in Microbiology ◽

10.3389/fmicb.2010.00130 ◽

2010 ◽

Vol 1 ◽

Cited By ~ 21

Author(s):

Maria Loza-Correa ◽

Laura Gomez-Valero ◽

Carmen Buchrieser

Keyword(s):

Circadian Clock ◽

Clock Proteins ◽

Circadian Clock Proteins

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Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation

Frontiers in Neuroscience ◽

10.3389/fnins.2021.647589 ◽

2021 ◽

Vol 15 ◽

Author(s):

Atsushige Ashimori ◽

Yasukazu Nakahata ◽

Toshiya Sato ◽

Yuichiro Fukamizu ◽

Takaaki Matsui ◽

...

Keyword(s):

Subcellular Localization ◽

Circadian Clock ◽

Posttranslational Modifications ◽

Cultured Cells ◽

Circadian Oscillation ◽

Clock Proteins ◽

Circadian Clock Proteins ◽

Circadian Physiology ◽

And Behavior ◽

Robust Systems

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.

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2PT229 The regulation of cyanobacterial circadian clock proteins KaiB-KaiC interaction by ATP(The 50th Annual Meeting of the Biophysical Society of Japan)

Seibutsu Butsuri ◽

10.2142/biophys.52.s143_5 ◽

2012 ◽

Vol 52 (supplement) ◽

pp. S143

Author(s):

Risa Mutoh ◽

Atsuhito Nishimura ◽

So Yasui ◽

Kiyoshi Onai ◽

Masahiro Ishiura

Keyword(s):

Circadian Clock ◽

Annual Meeting ◽

Clock Proteins ◽

Biophysical Society ◽

Circadian Clock Proteins

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CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock

Journal of Biological Rhythms ◽

10.1177/0748730419900868 ◽

2020 ◽

Vol 35 (3) ◽

pp. 227-234 ◽

Cited By ~ 1

Author(s):

Pyonghwa Kim ◽

Brianna Porr ◽

Tetsuya Mori ◽

Yong-Sung Kim ◽

Carl H. Johnson ◽

...

Keyword(s):

Circadian Clock ◽

Time Of Day ◽

Fine Tuning ◽

Circadian Oscillation ◽

Clock Proteins ◽

Entrainment Process ◽

Central Oscillator ◽

External Signals ◽

Full Synchronization

The circadian clock is a timekeeping system in most organisms that keeps track of the time of day. The rhythm generated by the circadian oscillator must be constantly synchronized with the environmental day/night cycle to make the timekeeping system truly advantageous. In the cyanobacterial circadian clock, quinone is a biological signaling molecule used for entraining and fine-tuning the oscillator, a process in which the external signals are transduced into biological metabolites that adjust the phase of the circadian oscillation. Among the clock proteins, the pseudo-receiver domain of KaiA and CikA can sense external cues by detecting the oxidation state of quinone, a metabolite that reflects the light/dark cycle, although the molecular mechanism is not fully understood. Here, we show the antagonistic phase shifts produced by the quinone sensing of KaiA and CikA. We introduced a new cyanobacterial circadian clock mixture that includes an input component in vitro. KaiA and CikA cause phase advances and delays, respectively, in this circadian clock mixture in response to the quinone signal. In the entrainment process, oxidized quinone modulates the functions of KaiA and CikA, which dominate alternatively at day and night in the cell. This in turn changes the phosphorylation state of KaiC—the central oscillator in cyanobacteria—ensuring full synchronization of the circadian clock. Moreover, we reemphasize the mechanistic input functionality of CikA, contrary to other reports that focus only on its output action.

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Relationship between HIF-1 and Circadian Clock Proteins in Obstructive Sleep Apnea Patients—Preliminary Study

Journal of Clinical Medicine ◽

10.3390/jcm9051599 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1599 ◽

Cited By ~ 4

Author(s):

Agata Gabryelska ◽

Marcin Sochal ◽

Szymon Turkiewicz ◽

Piotr Białasiewicz

Keyword(s):

Obstructive Sleep Apnea ◽

Sleep Apnea ◽

Circadian Clock ◽

Protein Level ◽

Hypoxia Inducible Factor ◽

Apnea Hypopnea Index ◽

Clock Proteins ◽

Obstructive Sleep ◽

Healthy Control ◽

Circadian Clock Proteins

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia and associated with the disruption of circadian rhythm. The study aimed to assess the relationship between hypoxia-inducible factor (HIF) subunits, circadian clock proteins, and polysomnography (PSG) variables, in healthy individuals and severe OSA patients. The study included 20 individuals, who underwent PSG and were divided into severe OSA group (n = 10; AHI ≥ 30) and healthy control (n = 10; AHI < 5) based on apnea-hypopnea index (AHI). All participants had their peripheral blood collected in the evening before and the morning after the PSG. HIF-1α, HIF-1β, BMAL1, CLOCK, CRY1, and PER1 protein concertation measurements were performed using ELISA. In a multivariate general linear model with the concentration of all circadian clock proteins as dependent variables, evening HIF-1α protein level was the only significant covariant (p = 0.025). Corrected models were significant for morning and evening PER1 (p = 0.008 and p = 0.006, respectively), evening (p = 0.043), and evening BMAL protein level (p = 0.046). In corrected models, evening HIF-1α protein level had an influence only on the evening PER1 protein level. Results suggest that OSA patients are at risk for developing circadian clock disruption. This process might be mediated by subunit α of HIF-1, as its increased protein level is associated with overexpression of circadian clock proteins.

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Synechocystis KaiC3 Displays Temperature- and KaiB-Dependent ATPase Activity and Is Important for Growth in Darkness

Journal of Bacteriology ◽

10.1128/jb.00478-19 ◽

2019 ◽

Vol 202 (4) ◽

Author(s):

Anika Wiegard ◽

Christin Köbler ◽

Katsuaki Oyama ◽

Anja K. Dörrich ◽

Chihiro Azai ◽

...

Keyword(s):

Atpase Activity ◽

Circadian Clock ◽

Atp Hydrolysis ◽

Synechococcus Elongatus ◽

Constant Darkness ◽

Content Type ◽

Clock Proteins ◽

Pcc 7942 ◽

Pcc 6803

ABSTRACT Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro. KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems. IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

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SnapShot: Circadian Clock Proteins

Cell ◽

10.1016/j.cell.2008.09.042 ◽

2008 ◽

Vol 135 (2) ◽

pp. 368-368.e1 ◽

Cited By ~ 13

Author(s):

Elizabeth E. Hamilton ◽

Steve A. Kay

Keyword(s):

Circadian Clock ◽

Clock Proteins ◽

Circadian Clock Proteins

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Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock

Methods and Protocols ◽

10.3390/mps2020042 ◽

2019 ◽

Vol 2 (2) ◽

pp. 42 ◽

Cited By ~ 4

Author(s):

Joel Heisler ◽

Archana Chavan ◽

Yong-Gang Chang ◽

Andy LiWang

Keyword(s):

Circadian Clock ◽

Real Time ◽

Fluorescence Anisotropy ◽

Polyacrylamide Gel Electrophoresis ◽

Live Cells ◽

Ex Post ◽

Clock Proteins ◽

Ex Post Facto ◽

Clock Reaction

Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.

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