Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.14952/seikagaku.2023.950604

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

Positron emission tomography (PET) is a powerful imaging tool that enables early in vivo detection of Alzheimer's disease (AD). For this purpose, various PET ligands have been developed to image β-amyloid and tau protein aggregates characteristically found in the brain of AD patients. In this study, we initiated to develop another type of PET ligand that targets protein kinase CK2 (formerly termed as casein kinase II), because its expression level is known to be altered in postmortem AD brains. CK2 is a serine/threonine protein kinase, an important component of cellular signaling pathways that control cellular degeneration. In AD, the CK2 level in the brain is thought to be elevated by its involvement in both phosphorylation of proteins such as tau and neuroinflammation. Decreased CK2 activity and expression levels lead to β-amyloid accumulation. In addition, since CK2 also contributes to the phosphorylation of tau protein, the expression level and activity of CK2 is expected to undergo significant changes during the progression of AD pathology. Furthermore, CK2 could act as a potential target for modulating the inflammatory response in AD. Therefore, PET imaging targeting CK2 expressed in the brain could be a useful another imaging biomarker for AD. We synthesized and radiolabeled a CK2 inhibitor, [11C]GO289, in high yields from its precursor and [11C]methyl iodide under basic conditions. On autoradiography, [11C]GO289 specifically bound to CK2 in both rat and human brain sections. On baseline PET imaging, this ligand entered and rapidly washed out of the rat brain with its peak activity rather being small (SUV < 1.0). However, on blocking, there was no detectable CK2 specific binding signal. Thus, [11C]GO289 may be useful in vitro but not so in vivo in its current formulation. The lack of detectable specific binding signal in the latter may be due to a relatively high component of nonspecific binding signal in the overall rather weak PET signal, or it may also be related to the known fact that ATP can competitively binds to subunits of CK2, reducing its availability for this ligand. In the future, it will be necessary for PET imaging of CK2 to try out different non-ATP competitive formulations of CK2 inhibitor that can also provide significantly higher in vivo brain penetration.

DOI： 10.1016/j.bmcl.2023.129327

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CK2 is a Ser/Thr protein kinase composed of two catalytic (α/α') subunits and a non-catalytic β-subunit dimer, whose activity is often abnormally high in cancer cells. The concept that CK2 may be dispensable for cell survival has been challenged by the finding that viable CK2α/α' knock-out myoblast clones still express small amounts of an N-terminally deleted α' subunit generated during the CRISPR/Cas9 procedure. Here we show that, although the overall CK2 activity of these CK2α(-/-)/Δα' (KO) cells is less than 10% compared to wild-type (WT) cells, the number of phosphosites with the CK2 consensus is comparable to that of WT cells. A more in-depth analysis, however, reveals that the two phosphoproteomes are not superimposable according to a number of criteria, notably a functional analysis of the phosphoproteome found in the two types of cells, and variable sensitivity of the phosphosites to two structurally unrelated CK2 inhibitors. These data support the idea that a minimal CK2 activity, as in KO cells, is sufficient to perform basic housekeeping functions essential for cell survival, but not to accomplish several specialized tasks required upon cell differentiation and transformation. From this standpoint, a controlled downregulation of CK2 would represent a safe and valuable anti-cancer strategy.

DOI： 10.1098/rsob.220220

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Background: Impairment of the circadian clock has been associated with numerous diseases, including sleep disorders and metabolic disease. Although small molecules that modulate clock function may form the basis of drug discovery of clock-related diseases, only a few compounds that selectively target core clock proteins have been identified. Three scaffolds were previously discovered as small-molecule activators of the clock protein Cryptochrome (CRY), and they have been providing powerful tools to understand and control the circadian clock system. Identifying new scaffolds will expand the possibilities of drug discovery. Methods: A methylbenzimidazole derivative TH401 identified from cell-based circadian screens was characterized. Effects of TH401 on circadian rhythms were evaluated in cellular assays. Functional assays and X-ray crystallography were used to elucidate the effects of the compound on CRY1 and CRY2 isoforms. Results: TH401 lengthened the period of circadian rhythms and stabilized both CRY1 and CRY2. The compound repressed Per2 reporter activity, which was reduced by Cry1 or Cry2 knockout and abolished by Cry1/Cry2 double knockout, indicating the dependence on CRY isoforms. Thermal shift assays showed slightly higher interaction of TH401 with CRY2 over CRY1. The crystal structure of CRY1 in complex with TH401 revealed a conformational change of the gatekeeper W399, which is involved in isoform-selectivity determination. Conclusions: The present study identified a new small molecule TH401 that targets both CRY isoforms. This compound has expanded the chemical diversity of CRY activators, and will ultimately aid in the development of therapeutics against circadian clock-related disorders.

DOI： 10.12688/f1000research.124658.1

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Authorship：Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：公益社団法人 日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu.60.212

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional-translational feedback loops (TTFLs). However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain (CTD) of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

DOI： 10.1093/pcp/pcac011

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cryptochromes (CRYs) are core components of the circadian feedback loop in mammals, which regulates circadian rhythmicity in a variety of physiological processes including sleep-wake cycles and metabolism. Dysfunction of CRY1 and CRY2 isoforms has been associated with a host of diseases, such as sleep phase disorder and metabolic diseases. Accumulating evidence for distinct roles of CRY1 and CRY2 has highlighted the need for CRY isoform-selective regulation; however, highly conserved sequences in CRY ligand-binding sites have hindered the design of isoform-selective compounds. Chemical biology approaches have been identifying small-molecule modulators of CRY proteins, which act in isoform-non-selective and also isoform-selective manners. In this review, we describe advances in our understanding of CRY isoform selectivity by comparing X-ray crystal structures of mammalian CRY isoforms in apo form and in complexes with compounds. We discuss how intrinsic conformational differences in identical residues of CRY1 and CRY2 contribute to unique interactions with different compound moieties for isoform selectivity.

DOI： 10.3389/fphys.2022.837280

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

There is increasing demand to control circadian clock functions in a conditional manner for deeper understanding of the circadian system as well as for potential treatment of clock-related diseases. Small-molecule compounds provide powerful tools to reveal novel functions of target proteins in the circadian clock mechanism, and can be great therapeutic candidates. Here we describe the detailed methods of measuring cellular circadian rhythms in a high-throughput manner for chemical screening to identify compounds that affect circadian rhythms by targeting clock-related proteins.

DOI： 10.1007/978-1-0716-2249-0_6

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Neuromethods  

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Language：English   Publishing type：Research paper (scientific journal)  

Circadian rhythm is an approximately 24 h endogenous biological rhythm. Chronic disruption of the circadian clock leads to an increased risk of diabetes, cardiovascular disease, and cancer. Hence, it is important to develop circadian clock modulators. Natural organisms are a good source of several medicines currently in use. Crude drugs used in Japanese traditional Kampo medicine or folk medicines are an excellent source for drug discovery. Furthermore, identifying new functions for existing drugs, known as the drug repositioning approach, is a popular and powerful tool. In this study, we screened 137 crude drug extracts to act as circadian clock modulators in human U2OS cells stably expressing the clock reporter Bmal1-dLuc, and approximately 12% of these modulated the circadian rhythm. We further examined the effects of several crude drugs in Rat-1 fibroblasts stably expressing Per2-luc, explant culture of lung from Per2::Luciferase knockin mice, and zebrafish larvae in vivo. Notably, more than half of the major ingredients of these crude drugs were reported to target AKT and its relevant signaling pathways. As expected, analysis of the major ingredients targeting AKT signaling confirmed the circadian clock-modulating effects. Furthermore, activator and inhibitor of AKT, and triple knockdown of AKT isoforms by siRNA also modulated the circadian rhythm. This study, by employing the drug repositioning approach, shows that Kampo medicines are a useful source for the identification of underlying mechanisms of circadian clock modulators and could potentially be used in the treatment of circadian clock disruption.

DOI： 10.1038/s41598-021-00499-w

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The circadian clock controls daily rhythms of various physiological processes, and impairment of its function causes many diseases including sleep disorders. Chemical compounds that regulate clock function are expected to be applied for treatment of circadian clock-related diseases. We previously identified small-molecule compounds KL001, KL101, and TH301 that lengthen the period of cellular circadian clock by directly targeting clock proteins Cryptochromes (CRYs) in mammals. KL001 targets both CRY1 and CRY2 isoforms, while KL101 and TH301 are isoform-selective compounds and require CRY C-terminal region for their effects. For further application of these compounds, the effects on locomotor activity rhythms at the organismal level need to be investigated. Here we used zebrafish larvae as an in vivo model system and found that KL001 lengthened the period of locomotor activity rhythms in a dose-dependent manner. In contrast, KL101 and TH301 showed no effect on the period. The amino acid sequences of CRY C-terminal regions are diverged in zebrafish and mammals, supporting the importance of this region for the effects of KL101 and TH301. This study demonstrated efficacy of CRY modulation for controlling circadian behavioral rhythms in organisms and suggested species-dependent differences in the effects of isoform-selective CRY-modulating compounds.

DOI： 10.1093/jb/mvab096

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Pharmaceutical Society of Japan  

The circadian clock is a biological system ubiquitous to almost all organisms on the earth, and controls daily rhythms in our body. Many physiological functions are rhythmically regulated by the cycles of expression, post-translational modification, and degradation of the clock proteins constituting the circadian clock. Disruption of circadian rhythms due to shift work and social jet lag is getting worse in modern society, and has been related with not only sleep disorders, but also various diseases, such as cancer and metabolic disorders. Small-molecule compounds targeting the clock-related proteins will provide a useful tool for uncovering the molecular mechanisms of circadian-related diseases and their treatments. In this article, we introduce the unique circadian clock modulating compounds and their potential for drug discovery.

DOI： 10.14894/medchem.31.2_62

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

CK2 (an acronym derived from the misnomer "casein kinase 2") denotes a ubiquitous, highly pleiotropic protein kinase which has been implicated in global human pathologies, with special reference to cancer. A large spectrum of fairly selective, cell permeable CK2 inhibitors are available, one of which, CX4945 is already in clinical trials for the treatment of neoplasia. Another recently developed CK2 inhibitor, GO289, displays in vitro potency and selectivity comparable to CX4945. Here the cellular efficiency of these two inhibitors has been evaluated by treating C2C12 myoblasts for 5 h with each of them at 4 μM concentration and running a quantitative phosphoproteomics analysis of phosphosites affected by the two compounds. A small but significant proportion of the quantified phosphosites is decreased by treatment with CX4945 and, even more with GO289. This figure substantially increases if a subset of quantified phosphosites conforming to the CK2 consensus (pS/pT-x-x-D/E/pS/pT) is considered. Also in this case GO289 is more effective than CX4945. By adopting stringent criteria two shortlists of 70 and 35 sites whose phosphorylation is decreased >50% by GO289 and CX4945, respectively, have been generated. All these phosphosites conform to the consensus of CK2 with just sporadic exceptions. Their WebLogos are indistinguishable from that of bona fide CK2 phosphosites and their Two-Sample Logos rule out any significant contribution of Pro-directed and basophilic protein kinases to their generation. To sum up, we can conclude that by treating C2C12 cells for 5 h with either CX4945 or GO289 off-target effects are negligible since almost all the phosphosites undergoing a substantial reduction are attributable to CK2, with a higher inhibitory efficacy displayed by GO289. CX4945 and GO289 provide highly selective tools to control the CK2-dependent phosphoproteome compared with previously developed CK2 inhibitors.

DOI： 10.1016/j.ejmech.2021.113217

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of bioactive compounds, such as known enzyme inhibitors. Until now, the development of photocontrolled protein kinase inhibitors proved to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase Iα (CKIα) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of the cis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKIα activity together with the accompanying modulation of cellular circadian rhythms in which CKIα is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a new potent reduced azopurine with a circadian period lengthening effect more pronounced than that of its parent molecule, longdaysin. Altogether, the results presented here highlight the challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications.

DOI： 10.1039/D1OB00014D

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

CiNii Research

Authorship：Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Viable clones of C2C12 myoblasts where both catalytic subunits of protein kinase CK2 had been knocked out by the CRISPR/Cas9 methodology have recently been generated, thus challenging the concept that CK2 is essential for cell viability. Here we present evidence that these cells are still endowed with a residual "CK2-like" activity that is able to phosphorylate Ser-13 of endogenous CDC37. Searching for a molecular entity accounting for such an activity we have identified a band running slightly ahead of CK2α' on SDS-PAGE. This band is not detectable by in-gel casein kinase assay but it co-immuno-precipitates with the β-subunit being downregulated by specific CK2α' targeting siRNA treatment. Its size and biochemical properties are consistent with those of CK2α' mutants deleted upstream of Glu-15 generated during the knockout process. This mutant sheds light on the role of the CK2 N-terminal segment as a regulator of activity and stability. Comparable cytotoxic efficacy of two selective and structurally unrelated CK2 inhibitors support the view that survival of CK2α/α'-/- cells relies on this deleted form of CK2α', whose discovery provides novel perspectives about the biological role of CK2.

DOI： 10.1016/j.bbrc.2020.07.112

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Cryptochrome 1 (CRY1) and CRY2 are core regulators of the circadian clock, and the development of isoform-selective modulators is important for the elucidation of their redundant and distinct functions. Here, we report the identification and functional characterization of a small-molecule modulator of the mammalian circadian clock that selectively controls CRY1. Cell-based circadian chemical screening identified a thienopyrimidine derivative KL201 that lengthened the period of circadian rhythms in cells and tissues. Functional assays revealed stabilization of CRY1 but not CRY2 by KL201. A structure-activity relationship study of KL201 derivatives in combination with X-ray crystallography of the CRY1-KL201 complex uncovered critical sites and interactions required for CRY1 regulation. KL201 bound to CRY1 in overlap with FBXL3, a subunit of ubiquitin ligase complex, and the effect of KL201 was blunted by knockdown of FBXL3. KL201 will facilitate isoform-selective regulation of CRY1 to accelerate chronobiology research and therapeutics against clock-related diseases.

DOI： 10.1016/j.chembiol.2020.05.008

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DOI： 10.1016/j.chempr.2020.08.011

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Pharmacological Society  

<p>In mammals, circadian rhythms are generated through transcriptional regulatory networks of the clock genes. To search for novel clock modifiers, we applied chemical biology approaches. From hundreds of thousands of small molecules with diverse structure, we identified a number of compounds that potently change the period of the circadian clock in human cells. Among the period lengthening compounds, we previously discovered the first small molecule targeting the core clock protein CRY. The compound KL001 interacts with FAD-binding pocket of CRY and inhibits FBXL3-dependent degradation. By analyzing KL001 derivatives, we found 10 times more potent compound KL044. KL001 and KL044 share carbazole group and act on both CRY1 and CRY2. We further identified novel period lengthening compounds KL101 and TH301 that do not have carbazole group. Surprisingly, we discovered that KL101 is selective against CRY1 while TH301 shows much higher effect on CRY2. To understand molecular basis of the CRY1/CRY2 selectivity, we determined the X-ray crystal structures of CRY1-KL101, CRY1-TH301, CRY2-TH301, and CRY1-KL044 complexes. In this presentation, I will discuss these unique compounds that will enable atomic-level dissection of the functional difference between CRY1 and CRY2 proteins and their selective manipulation.</p>

DOI： 10.1254/jpssuppl.93.0_1-s12-3

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Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.

DOI： 10.1016/j.bbamcr.2020.118807

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Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

DOI： 10.7554/eLife.55275

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Daily physiological rhythms are regulated by the body's internal timekeeper known as the circadian clock. Expression, post-translational modification, and degradation of clock proteins constituting the circadian clock are precisely controlled in a rhythmic manner. Perturbation of these processes by nature and nurture results in physiological dysfunction and diseases. Small-molecule modulators of clock or clock-related proteins can adjust clock functions, and thus represent a promising method of therapeutic treatment for a range of clock-related diseases. In this review, we will introduce the identification and development of small-molecule compounds that target clock proteins, as well as X-ray crystal structures of protein-compound complexes that facilitate the understanding of clock protein regulation and drug derivatization. Furthermore, we describe the effects of these compounds in a diseased setting and discuss the therapeutic potential of clock modulators.

DOI： 10.1016/j.jmb.2020.01.003

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CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

DOI： 10.1038/s41589-020-0505-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.

DOI： 10.1038/s41598-019-48914-7

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Other Link： http://orcid.org/0000-0003-4876-3608

Language：English   Publishing type：Research paper (scientific journal)  

The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

DOI： 10.1073/pnas.1903357116

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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.

DOI： 10.1126/sciadv.aau9060

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Circadian clocks, biological timekeepers that are present in almost every cell of our body, are complex systems whose disruption is connected to various diseases. Controlling cellular clock function with high temporal resolution in an inducible manner would yield an innovative approach for the circadian rhythm regulation. In the present study, we present structure-guided incorporation of photoremovable protecting groups into a circadian clock modifier, longdaysin, which inhibits casein kinase I (CKI). Using photodeprotection by UV or visible light (400 nm) as the external stimulus, we have achieved quantitative and light-inducible control over the CKI activity accompanied by an accurate regulation of circadian period in cultured human cells and mouse tissues, as well as in living zebrafish. This research paves the way for the application of photodosing in achieving precise temporal control over the biological timing and opens the door for chronophotopharmacology to deeper understand the circadian clock system.

DOI： 10.1021/jacs.9b05445

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

E2 conjugating enzymes are the key catalytic actors in the transfer of ubiquitin, SUMO, and other ubiquitin-like modifiers to their substrate proteins. Their high rates of transfer and promiscuous binding complicate studies of their interactions and binding partners. To access specific, covalently linked conjugates of the SUMO E2 conjugating enzyme Ubc9, we prepared synthetic variants bearing site-specific non-native modifications including the following: (1) replacement of Cys93 to 2,3-diaminopropionic acid to form the amide-linked stable E2-SUMO conjugate, which is known to have high affinity for E3 ligases; (2) a photoreactive group (diazirine) to trap E3 ligases upon UV irradiation; and (3) an N-terminal biotin for purification and detection. To construct these Ubc9 variants in a flexible, convergent manner, we combined the three leading methods: native chemical ligation (NCL), α-ketoacid-hydroxylamine (KAHA) ligation, and serine/threonine ligation (STL). Using the synthetic proteins, we demonstrated the selective formation of Ubc9-SUMO conjugates and the trapping of an E3 ligase (RanBP2) to form the stable, covalently linked SUMO1-Ubc9-RanBP2 ternary complex. The powerful combination of ligation methods-which minimizes challenges of functional group manipulations-will enable chemical probes based on E2 conjugating enzymes to trap E3 ligases and facilitate the synthesis of other protein classes.

DOI： 10.1021/jacs.9b06820

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Circadian rhythms are controlled by transcriptional feedback loops of clock genes and proteins. The stability of clock proteins is regulated by post-translational modification, such as phosphorylation by kinases. In particular, casein kinase I (CKI) phosphorylates the PER protein to regulate proteasomal degradation and nuclear localization. Therefore, CKI inhibition can modulate mammalian circadian rhythms. In the present study, we have developed novel CKIα and CKIδ dual inhibitors by extensive structural modification of N9 and C2 position of longdaysin. We identified NCC007 that showed stronger period effects (0.32 μM for 5 h period lengthening) in a cell-based circadian assay. The following in vitro kinase assay showed that NCC007 inhibited CKIα and CKIδ with an IC50 of 1.8 and 3.6 μM. We further demonstrated that NCC007 lengthened the period of mouse behavioral rhythms in vivo. Thus, NCC007 is a valuable tool compound to control circadian rhythms through CKI inhibition.

DOI： 10.1021/acs.jmedchem.8b01541

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Inc.  

Protein kinase, casein kinase II (CK2), is ubiquitously expressed and highly conserved protein kinase that shows constitutive activity. It phosphorylates a diverse set of proteins and plays crucial role in several cellular processes. The catalytic subunit of this enzyme (CK2α) shows remarkable flexibility as evidenced in numerous crystal structures determined till now. Here, using analysis of multiple crystal structures and long timescale molecular dynamics simulations, we explore the conformational flexibility of CK2α. The enzyme shows considerably higher flexibility in the solution as compared to that observed in crystal structure ensemble. Multiple conformations of hinge region, located near the active site, were observed during the dynamics. We further observed that among these multiple conformations, the most populated conformational state was inadequately represented in the crystal structure ensemble. The catalytic spine, was found to be less dismantled in this state as compared to the “open” hinge/αD state crystal structures. The comparison of dynamics in unbound (Apo) state and inhibitor (CX4945) bound state exhibits inhibitor induced suppression in the overall dynamics of the enzyme. This is especially true for functionally important glycine-rich loop above the active site. Together, this work gives novel insights into the dynamics of CK2α in solution and relates it to the function. This work also explains the effect of inhibitor on the dynamics of CK2α and paves way for development of better inhibitors.

DOI： 10.1002/prot.25444

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Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly transrepresses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed cooccupancy of HNF4A and CLOCK:BMAL1 at a wide array of metabolic genes involved in lipid, glucose, and amino acid homeostasis. Taken together, we establish that HNF4A defines a feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways.

DOI： 10.1073/pnas.1816411115

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Mammals receive light information through the eyes, which perform two major functions: image forming vision to see objects and non-image forming adaptation of physiology and behavior to light. Cone and rod photoreceptors form images and send the information via retinal ganglion cells to the brain for image reconstruction. In contrast, nonimage-forming photoresponses vary widely from adjustment of pupil diameter to adaptation of the circadian clock. nonimage-forming responses are mediated by retinal ganglion cells expressing the photopigment melanopsin. Melanopsin-expressing cells constitute 1-2% of retinal ganglion cells in the adult mammalian retina, are intrinsically photosensitive, and integrate photic information from rods and cones to control nonimage-forming adaptation. Action spectra of ipRGCs and of melanopsin photopigment peak around 480 nm blue light. Understanding melanopsin function lets us recognize considerable physiological effects of blue light, which is increasingly important in our modern society that uses light-emitting diode. Misalignment of circadian rhythmicity is observed in numerous conditions, including aging, and is thought to be involved in the development of age-related disorders, such as depression, diabetes, hypertension, obesity, and cancer. The appropriate regulation of circadian rhythmicity by proper lighting is therefore essential. This perspective introduces the potential risks of excessive blue light for human health through circadian rhythm disruption and sleep deprivation. Knowing the positive and negative aspects, this study claims the importance of being exposed to light at optimal times and intensities during the day, based on the concept of the circadian clock, ultimately to improve quality of life to have a healthy and longer life.

DOI： 10.1038/s41514-017-0010-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERB alpha, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERB alpha-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERB alpha is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERB alpha repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition.

DOI： 10.1016/j.cell.2016.05.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The synthesis and functional analysis of KL001 derivatives, which are modulators of the mammalian circadian clock, are described. By using cutting-edge C-H activation chemistry, a focused library of KL001 derivatives was rapidly constructed, which enabled the identification of the critical sites on KL001 derivatives that induce a rhythm-changing activity along with the components that trigger opposite modes of action. The first period-shortening molecules that target the cryptochrome (CRY) were thus discovered. Detailed studies on the effects of these compounds on CRY stability implicate the existence of an as yet undiscovered regulatory mechanism.

DOI： 10.1002/anie.201502942

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Small-molecule probes have been playing prominent roles in furthering our understanding of the molecular underpinnings of the circadian clock. We previously discovered a carbazole derivative, KL001 (N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)-N-(furan-2-ylmethyl)methanesulfonamide), as a stabilizer of the clock protein cryptochrome (CRY). Herein we describe an extensive structure-activity relationship analysis of KL001 derivatives leading to the development of a highly active derivative: 2-(9H-carbazol-9-yl)-N-(2-chloro-6-cyanophenyl)acetamide (KL044). Subsequent 3D-QSAR analysis identified critical features of KL001 derivatives and provided a molecular-level understanding of their interaction with CRY. The electron-rich carbazole, amide/hydroxy linker, sulfonyl group, and electron-withdrawing nitrile moieties contribute to greater biological activity. The hydrogen bonding interactions with Ser394 and His357 as well as stronger CH- interactions with Trp290 make KL044 a better binder than KL001. KL044 lengthened the circadian period, repressed Per2 activity, and stabilized CRY in reporter assays with roughly tenfold higher potency than KL001. Altogether, KL044 is a powerful chemical tool to control the function of the circadian clock through its action on CRY.

DOI： 10.1002/cmdc.201500260

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Methods in Enzymology  

Chemical biology or chemical genetics has emerged as an interdisciplinary research area applying chemistry to understand biological systems. The development of combinatorial chemistry and high-throughput screening technologies has enabled large-scale investigation of the biological activities of diverse small molecules to discover useful chemical probes. This approach is applicable to the analysis of the circadian clock mechanisms through cell-based assays to monitor circadian rhythms using luciferase reporter genes. We and others have established cell-based high-throughput circadian assays and have identified a variety of novel small-molecule modulators of the circadian clock by phenotype-based screening of hundreds of thousands of compounds. The results demonstrated the effectiveness of chemical biology approaches in clock research field. This technique will become more and more common with propagation of high-throughput screening facilities. This chapter describes assay development, screening setups, and their optimization for successful screening campaigns.

DOI： 10.1016/bs.mie.2014.10.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The circadian clock is finely regulated by posttranslational modifications of clock components. Mouse CRY2, a critical player in the mammalian clock, is phosphorylated at Ser557 for proteasome-mediated degradation, but its in vivo role in circadian organization was not revealed. Here, we generated CRY2(S557A) mutant mice, in which Ser557 phosphorylation is specifically abolished. The mutation lengthened free-running periods of the behavioral rhythms and PER2:: LUC bioluminescence rhythms of cultured liver. In livers from mutant mice, the nuclear CRY2 level was elevated, with enhanced PER2 nuclear occupancy and suppression of E-box-regulated genes. Thus, Ser557 phosphorylation-dependent regulation of CRY2 is essential for proper clock oscillation in vivo.

DOI： 10.1128/MCB.00711-14

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Posttranslational regulation of clock proteins is an essential part of mammalian circadian rhythms, conferring sensitivity to metabolic state and offering promising targets for pharmacological control. Two such regulators, casein kinase 1 (CKI) and F-box and leucine-rich repeat protein 3 (FBXL3), modulate the stability of closely linked core clock proteins period (PER) and cryptochrome (CRY), respectively. Inhibition of either CKI or FBXL3 leads to longer periods, and their effects are independent despite targeting proteins with similar roles in clock function. A mechanistic understanding of this independence, however, has remained elusive. Our analysis of cellular circadian clock gene reporters further differentiated between the actions of CKI and FBXL3 by revealing opposite amplitude responses from each manipulation. To understand the functional relationship between the CKI-PER and FBXL3-CRY pathways, we generated robust mechanistic predictions by applying a bootstrap uncertainty analysis to multiple mathematical circadian models. Our results indicate that CKI primarily regulates the accumulating phase of the PER-CRY repressive complex by controlling the nuclear import rate, whereas FBXL3 separately regulates the duration of transcriptional repression in the nucleus. Dynamic simulations confirmed that this spatiotemporal separation is able to reproduce the independence of the two regulators in period regulation, as well as their opposite amplitude effect. As a result, this study provides further insight into the molecular clock machinery responsible for maintaining robust circadian rhythms.

DOI： 10.1073/pnas.1323618111

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The central and peripheral nervous systems have the capacity of synthesizing steroids de novo from cholesterol, the so-called 'neurosteroids'. De novo synthesis of neurosteroids from cholesterol appears to be a conserved property across the subphylum vertebrata. Until recently, it was generally believed that neurosteroids are produced in neurons and glial cells in the central and peripheral nervous systems. However, our recent studies on birds have demonstrated that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol. 7 alpha-Hydroxypregnenolone is a major pineal neurosteroid that stimulates locomotor activity of juvenile birds, connecting light-induced gene expression with locomotion. The other major pineal neurosteroid allopregnanolone is involved in Purkinje cell survival by suppressing the activity of caspase-3, a crucial mediator of apoptosis during cerebellar development. This review is an updated summary of the biosynthesis and biological actions of pineal neurosteroids. Copyright (C) 2013 S. Karger AG, Basel

DOI： 10.1159/000353782

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De novo neurosteroidogenesis from cholesterol occurs in the brain of various avian species. However, the biosynthetic pathways leading to the formation of neurosteroids are still not completely elucidated. We have recently found that the avian brain produces 7α-hydroxypregnenolone, a novel bioactive neurosteroid that stimulates locomotor activity. Until recently, it was believed that neurosteroids are produced in neurons and glial cells in the central and peripheral nervous systems. However, our recent studies on birds have demonstrated that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol. 7α-Hydroxypregnenolone is a major pineal neurosteroid that stimulates locomotor activity of juvenile birds, connecting light-induced gene expression with locomotion. The other major pineal neurosteroid allopregnanolone is involved in Purkinje cell survival during development. This paper highlights new aspects of neurosteroid synthesis and actions in birds.

DOI： 10.4137/JEN.S11148

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The circadian clock co-ordinates physiology and behavior with the day/night cycle. It consists of a transcriptional-translational feedback loop that generates self-sustained oscillations in transcriptional activity with a roughly 24 h period via E-box enhancer elements. Numerous in vivo aspects of core clock feedback loop function are still incompletely understood, including its maturation during development, tissue-specific activity and perturbation in disease states. Zebrafish are promising models for biomedical research due to their high regenerative capacity and suitability for in vivo drug screens, and transgenic zebrafish lines are valuable tools to study transcriptional activity in vivo during development.
To monitor the activity of the core clock feedback loop in vivo, we created a transgenic zebrafish line expressing a luciferase reporter gene under the regulation of a minimal promoter and four E-boxes. This Tg(4xE-box:Luc) line shows robust oscillating reporter gene expression both under light-dark cycles and upon release into constant darkness. Luciferase activity starts to oscillate during the first days of development, indicating that the core clock loop is already functional at an early stage. To test whether the Tg(4xE-box:Luc) line could be used in drug screens aimed at identifying compounds that target the circadian clock in vivo, we examined drug effects on circadian period. We were readily able to detect period changes as low as 0.7 h upon treatment with the period-lengthening drugs lithium chloride and longdaysin in an assay set-up suitable for large-scale screens. Reporter gene mRNA expression is also detected in the adult brain and reveals differential clock activity across the brain, overlapping with endogenous clock gene expression. Notably, core clock activity is strongly correlated with brain regions where neurogenesis takes place and can be detected in several types of neural progenitors.
Our results demonstrate that the Tg(4xE-box:Luc) line is an excellent tool for studying the regulation of the circadian clock and its maturation in vivo and in real time. Furthermore, it is highly suitable for in vivo screens targeting the core clock mechanism that take into account the complexity of an intact organism. Finally, it allows mapping of clock activity in the brain of a vertebrate model organism with prominent adult neurogenesis and high regeneration capacity. (c) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2013.04.035

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Impairment of the circadian clock has been associated with numerous disorders, including metabolic disease. Although small molecules that modulate clock function might offer therapeutic approaches to such diseases, only a few compounds have been identified that selectively target core clock proteins. From an unbiased cell-based circadian phenotypic screen, we identified KL001, a small molecule that specifically interacts with cryptochrome (CRY). KL001 prevented ubiquitin-dependent degradation of CRY, resulting in lengthening of the circadian period. In combination with mathematical modeling, our studies using KL001 revealed that CRY1 and CRY2 share a similar functional role in the period regulation. Furthermore, KL001-mediated CRY stabilization inhibited glucagon-induced gluconeogenesis in primary hepatocytes. KL001 thus provides a tool to study the regulation of CRY-dependent physiology and aid development of clock-based therapeutics of diabetes.

DOI： 10.1126/science.1223710

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

A high-throughput cell-based screen identified a benzothiazole analogue, LH846, which induces period lengthening of the circadian rhythm. Affinity chromatography coupled with mass spectrometry and genomic analysis identified protein kinase CKIδ as the biological target of LH846 (see picture).

DOI： 10.1002/anie.201103915

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by &gt;2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF) 1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7 alpha-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7 alpha-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-day-dependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.

DOI： 10.1073/pnas.1015959108

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis1. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)-two key transcriptional regulators of this process(2). Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment(3-6). Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)alpha. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.

DOI： 10.1038/nm.2214

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding. In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts. We previously demonstrated that glucose treatment of rat-1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor. Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day/night transition and midnight, respectively. In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site. The GC box/TIEG1-mediated repression of Bmal1 promoter was additive to RORE-dependent repression by REV-ERB alpha, a well-known repressor of Bmal1 gene. In cell-based real-time assay, siRNA-mediated knock-down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1-luciferase and Per2-luciferase reporters. These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box-mediated regulation of Bmal1 transcription.

DOI： 10.1111/j.1365-2443.2009.01371.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of similar to 120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKI delta, CKI alpha, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKI delta, CKI alpha, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKI alpha in the clock mechanism and found that CKI alpha-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKI delta. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKI alpha as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.

DOI： 10.1371/journal.pbio.1000559

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Circadian molecular oscillation is generated by a transcription/translation-based feedback loop in which CRY proteins play critical roles as potent inhibitors for E-box-dependent clock gene expression. Although CRY2 undergoes rhythmic phosphorylation in its C-terminal tail, structurally distinct from the CRY1 tail, little is understood about how protein kinase(s) controls the CRY2-specific phosphorylation and contributes to the molecular clockwork. Here we found that Ser557 in the C-terminal tail of CRY2 is phosphorylated by DYRK1A as a priming kinase for subsequent GSK-3 beta (glycogen synthase kinase 3 beta)-mediated phosphorylation of Ser553, which leads to proteasomal degradation of CRY2. In the mouse liver, DYRK1A kinase activity toward Ser557 of CRY2 showed circadian variation, with its peak in the accumulating phase of CRY2 protein. Knockdown of Dyrk1a caused abnormal accumulation of cytosolic CRY2, advancing the timing of a nuclear increase of CRY2, and shortened the period length of the cellular circadian rhythm. Expression of an S557A/S553A mutant of CRY2 phenocopied the effect of Dyrk1a knockdown in terms of the circadian period length of the cellular clock. DYRK1A is a novel clock component cooperating with GSK-3 beta and governs the Ser557 phosphorylation-triggered degradation of CRY2.

DOI： 10.1128/MCB.01047-09

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.

DOI： 10.1016/j.cell.2009.08.031

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Most organisms exhibit daily changes in physiology and metabolism under the control of a cell-autonomous circadian clock. In the core clock mechanism, clock genes form a transcription factor network to generate circadian rhythms of gene expression. Clock protein phosphorylation and histone modifications are also important for the clock regulation. Pharmacological approaches have been making significant contributions to the clock research, for example, in characterizing the roles of protein kinases CKI delta, CKI epsilon, CK2, and GSK-3 beta. Recently, high-throughput circadian functional assays have been established. Chemical biology approaches utilizing high-throughput compound screening together with RNAi-based genomic screening will open a new way for the circadian clock field. Finding a set of compounds that potently affect the clock function will lead to the identification of novel clock components and form the basis for therapeutic strategies directed toward circadian disorders.

DOI： 10.1016/j.chembiol.2009.09.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The circadian clock controls daily oscillations of gene expression at the cellular level. We report the development of a high-throughput circadian functional assay system that consists of luminescent reporter cells, screening automation, and a data analysis pipeline. We applied this system to further dissect the molecular mechanisms underlying the mammalian circadian clock using a chemical biology approach. We analyzed the effect of 1,280 pharmacologically active compounds with diverse structures on the circadian period length that is indicative of the core clock mechanism. Our screening paradigm identified many compounds previously known to change the circadian period or phase, demonstrating the validity of the assay system. Furthermore, we found that small molecule inhibitors of glycogen synthase kinase 3 (GSK-3) consistently caused a strong short period phenotype in contrast to the well-known period lengthening by lithium, another presumed GSK-3 inhibitor. siRNA-mediated knockdown of GSK-3 beta also caused a short period, confirming the phenotype obtained with the small molecule inhibitors. These results clarify the role of GSK-3 beta in the period regulation of the mammalian clockworks and highlight the effectiveness of chemical biology in exploring unidentified mechanisms of the circadian clock.

DOI： 10.1073/pnas.0811410106

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The circadian clock is reset by external time cues for synchronization to environmental changes(1). In mammals, the light-input signalling pathway mediated by Per gene induction has been extensively studied(2,3). On the other hand, little is known about resetting mechanisms that are independent of Per induction(4-6). Here we show that activation of activin receptor-like kinase (ALK), triggered by TGF-beta, activin or alkali signals, evoked resetting of the cellular clock independently of Per induction. The resetting was mediated by an immediate-early induction of Dec1, a gene whose physiological role in the function of the circadian clock has been unclear. Acute Dec1 induction was a prerequisite for ALK-mediated resetting and upregulation was dependent on SMAD3, which was phosphorylated for activation in response to the resetting stimuli. Intraperitoneal injection of TGF-beta into wild-type or Dec1-deficient mice demonstrated that Dec1 has an essential role in phase-shift of clock gene expression in the kidney and adrenal gland. These results indicate that ALK-SMAD3-Dec1 signalling provides an input pathway in the mammalian molecular clock.

DOI： 10.1038/ncb1806

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The circadian clock in the retina regulates a variety of physiological phenomena such as disc shedding and melatonin release. Although these events are critical for retinal functions, it is almost unknown how the circadian clock controls the physiological rhythmicity. To gain insight into the processes, we performed a proteomic analysis using 2-DE to find proteins whose levels show circadian changes. Among 415 retinal protein spots, 11 protein spots showed circadian rhythmicity in their intensities. We performed MALDI-TOF MS and NanoLC-MS/MS analyses and identified proteins contained in the 11 spots. The proteins were related to vesicular transport, calcium-binding, protein degradation, metabolism, RNA-binding, and protein foldings, suggesting the clock-regulation of neurotransmitter release, transportation of the membrane proteins, calcium-binding capability, and so on. We also found a rhythmic phosphorylation of N-ethylmaleimide-sensitive fusion protein and identified one of the amino acid residues modified by phosphorylation. These findings provide a new perspective on the relationship between the physiological functions of the retina and the circadian clock system.

DOI： 10.1002/pmic.200700272

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3 beta (GSK-3 beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.

DOI： 10.1080/07420520500464478

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557- phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by similar to 4 h in both light-dark cycle and constant dark conditions. The Ser-557- phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3 beta (GSK-3 beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3 beta at Ser-9, which negatively regulates the kinase activity, GSK-3 beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.

DOI： 10.1074/jbc.M506225200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Almost all organisms on earth exhibit diurnal rhythms in physiology and behavior under the control of autonomous time-measuring system called circadian clock. The circadian clock is generally reset by environmental time cues, such as light, in order to synchronize with the external 24-h cycles. In mammals, the core oscillator of the circadian clock is composed of transcription/translation-based negative feedback loops regulating the cyclic expression of a limited number of clock genes (such as Per, Cry, Bmal1, etc.) and hundreds of output genes in a well-concerted manner. The central clock controlling the behavioral rhythm is localized in the hypothalamic suprachiasmatic nucleus (SCN), and peripheral clocks are present in other various tissues. The phase of the central clock is amenable to ambient light signal captured by the visual rod-cone photoreceptors and non-visual melanopsin in the retina. These light signals are transmitted to the SCN through the retinohypothalamic tract, and transduced therein by mitogen-activated protein kinase and other signaling molecules to induce Per gene expression, which eventually elicits phase-dependent phase shifts of the clock. The central clock controls peripheral clocks directly and indirectly by virtue of neural, humoral, and other signals in a coordinated manner. The change in feeding time resets the peripheral clocks in a SCN-independent manner, possibly by food metabolites and body temperature rhythms. In this article, we will provide an overview of recent molecular and genetic studies on the resetting mechanism of the central and peripheral circadian clocks in mammals.

DOI： 10.2108/zsj.21.359

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Extracellular signal-regulated kinase (ERK) and p38 are members of the mitogen-activated protein kinase (MAPK) family, and in some cases these kinases serve for closely related cellular functions within a cell. In a wide range of animal clock structures, ERK plays an important role in the circadian time-keeping mechanism. Here we found that immunoreactivity to p38 protein was uniformly distributed among cells in the chick pineal gland. On the other hand, a constant level of activated p38 was detected over the day, predominantly in the follicular and parafollicular pinealocytes that are potential circadian clock-containing cells. Chronic application of SB203580, a selective and reversible inhibitor of p38, to the cultured chick pineal cells markedly lengthened the period of the circadian rhythm of the melatonin release (up to 28.7 h). Noticeably, despite no significant temporal change of activated p38 level, a 4-h pulse treatment with SB203580 delayed the phase of the rhythm only when delivered during the subjective day. These results indicate a time-of-day-specific role of continuously activated p38 in the period length regulation of the chick pineal clock and suggest temporally separated regulation of the clock by two MAPKs, nighttime-activated ERK and daytime-working p38.

DOI： 10.1074/jbc.M212726200

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PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S11_2

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

In mammals, peripheral circadian clocks are present in most tissues, but little is known about how these clocks are synchronized with the ambient 24-h cycles. By using rat-1 fibroblasts, a model cell system of the peripheral clock, we found that an exchange of the culture medium triggered circadian gene expression that was preceded by slow down-regulation of Per1 and Per2 mRNA levels. This profile contrasts to the immediate up-regulation of these genes often observed for clock resetting. The screening of factor(s) responsible for the down-regulation revealed glucose as a key component triggering the circadian rhythm. The requirement of both glucose metabolism and RNA/protein synthesis for the down-regulation suggests the involvement of gene(s) immediately up-regulated by glucose metabolism. An analysis with high density oligonucleotide microarrays identified &gt;100 glucose-regulated genes. We found among others immediately up-regulated genes encoding transcriptional regulators TIEG1, VDUP1, and HES1, in addition to cooperatively regulated genes that are associated with cholesterol biosynthesis and cell cycle. The immediate up-regulation of Tieg1 and Vdup1 expression was dependent on glucose metabolism but not on protein synthesis, suggesting that the transcriptional regulators mediate the glucose-induced down-regulation of Per1 and Per2 expression. These results illustrate a novel mode of peripheral clock resetting by external glucose, a major food metabolite.

DOI： 10.1074/jbc.M206233200

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Background: In a transcription/translation-based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1-CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized.
Results: We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1-cCLOCK and cBMAL2-cCLOCK heteromers. Both of the cBMAL-cCLOCK heteromers activated E-box element-dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co-expression of cCLOCK, cBMAL1 and cBMAL2 co-operatively activated E-box element-dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over-expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production.
Conclusion: The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E-box-dependent transcriptional regulation in the circadian clock system.

DOI： 10.1046/j.1365-2443.2001.00462.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.

DOI： 10.1093/oxfordjournals.jbchem.a002836

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Scopus

Language：Japanese   Publisher：(公社)日本生化学会  

Language：Japanese  

CiNii Books

Language：Japanese   Publisher：(公社)日本生化学会  

Language：English   Publisher：Zoological Society of Japan  

CiNii Books

Other Link： http://id.nii.ac.jp/1141/00035547/