Death of a Protein: The Role of E3 Ubiquitin Ligases in Circadian Rhythms of Mice and Flies.

Circadian clocks evolved to enable organisms to anticipate and prepare for periodic environmental changes driven by the day-night cycle. This internal timekeeping mechanism is built on autoregulatory transcription-translation feedback loops that control the rhythmic expression of core clock genes and their protein products. The levels of clock proteins rise and ebb throughout a 24-h period through their rhythmic synthesis and destruction. In the ubiquitin-proteasome system, the process of polyubiquitination, or the covalent attachment of a ubiquitin chain, marks a protein for degradation by the 26S proteasome. The process is regulated by E3 ubiquitin ligases, which recognize specific substrates for ubiquitination. In this review, we summarize the roles that known E3 ubiquitin ligases play in the circadian clocks of two popular model organisms: mice and fruit flies. We also discuss emerging evidence that implicates the N-degron pathway, an alternative proteolytic system, in the regulation of circadian rhythms. We conclude the review with our perspectives on the potential for the proteolytic and non-proteolytic functions of E3 ubiquitin ligases within the circadian clock system.

Melatonin effects on bone: Implications for use as a therapy for managing bone loss.

Melatonin is the primary circadian output signal from the brain and is mainly synthesized in pinealocytes. The rhythm and secretion of melatonin are under the control of an endogenous oscillator located in the SCN or the master biological clock. Disruptions in circadian rhythms by shift work, aging, or light at night are associated with bone loss and increased fracture risk. Restoration of nocturnal melatonin peaks to normal levels or therapeutic levels through timed melatonin supplementation has been demonstrated to provide bone-protective actions in various models. Melatonin is a unique molecule with diverse molecular actions targeting melatonin receptors located on the plasma membrane or mitochondria or acting independently of receptors through its actions as an antioxidant or free radical scavenger to stimulate osteoblastogenesis, inhibit osteoclastogenesis, and improve bone density. Its additional actions on entraining circadian rhythms and improving quality of life in an aging population coupled with its safety profile make it an ideal therapeutic candidate for protecting against bone loss in susceptible populations. The intent of this review is to provide a focused discussion on bone loss and disorders of the bone as it relates to melatonin and conditions that modify melatonin levels with the hope that future therapies include those that include melatonin and correct those factors that modify melatonin levels like circadian disruption.

Recent advances in modulators of circadian rhythms: an update and perspective.

Circadian rhythm is a universal life phenomenon that plays an important role in maintaining the multiple physiological functions and regulating the adaptability to internal and external environments of flora and fauna. Circadian alignment in humans has the greatest effect on human health, and circadian misalignment is closely associated with increased risk for metabolic syndrome, cardiovascular diseases, neurological diseases, immune diseases, cancer, sleep disorders, and ophthalmic diseases. The recent description of clock proteins and related post-modification targets was involved in several diseases, and numerous lines of evidence are emerging that small molecule modulators of circadian rhythms can be used to rectify circadian disorder. Herein, we attempt to update the disclosures about the modulators targeting core clock proteins and related post-modification targets, as well as the relationship between circadian rhythm disorders and human health as well as the therapeutic role and prospect of these small molecule modulators in circadian rhythm related disease.

Atomic force microscopy analysis of SasA-KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria.

The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC-binding EnvZ-like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA3mer ) and occasionally as a hexamer (SasA6mer ) in vitro. Previously, the molecular mass of the SasA-KaiCDD complex, where KaiCDD is a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel-filtration chromatography to be larger than 670 kDa. This value disagrees with the theoretical estimation of 480 kDa for a SasA3mer -KaiC hexamer (KaiC6mer ) complex with a 1:1 molecular ratio. To clarify the structure of the SasA-KaiC complex, we analyzed KaiCDD with 0.1 mmol/L ATP and 5 mmol/L MgCl2 (Mg-ATP), SasA and a mixture containing SasA and KaiCDD6mer with Mg-ATP by atomic force microscopy (AFM). KaiCDD images were classified into two types with height distribution corresponding to KaiCDD monomer (KaiCDD1mer ) and KaiCDD6mer , respectively. SasA images were classified into two types with height corresponding to SasA3mer and SasA6mer , respectively. The AFM images of the SasA-KaiCDD mixture indicated not only KaiCDD1mer , KaiCDD6mer , SasA3mer and SasA6mer , but also wider area "islands," suggesting the presence of a polymerized form of the SasA-KaiCDD complex.

G Protein-Coupled Receptor Systems as Crucial Regulators of DNA Damage Response Processes.

G protein-coupled receptors (GPCRs) and their associated proteins represent one of the most diverse cellular signaling systems involved in both physiological and pathophysiological processes. Aging represents perhaps the most complex biological process in humans and involves a progressive degradation of systemic integrity and physiological resilience. This is in part mediated by age-related aberrations in energy metabolism, mitochondrial function, protein folding and sorting, inflammatory activity and genomic stability. Indeed, an increased rate of unrepaired DNA damage is considered to be one of the 'hallmarks' of aging. Over the last two decades our appreciation of the complexity of GPCR signaling systems has expanded their functional signaling repertoire. One such example of this is the incipient role of GPCRs and GPCR-interacting proteins in DNA damage and repair mechanisms. Emerging data now suggest that GPCRs could function as stress sensors for intracellular damage, e.g., oxidative stress. Given this role of GPCRs in the DNA damage response process, coupled to the effective history of drug targeting of these receptors, this suggests that one important future activity of GPCR therapeutics is the rational control of DNA damage repair systems.

Circadian Activators Are Expressed Days before They Initiate Clock Function in Late Pacemaker Neurons from Drosophila.

Circadian pacemaker neurons in the Drosophila brain control daily rhythms in locomotor activity. These pacemaker neurons can be subdivided into early or late groups depending on whether rhythms in period (per) and timeless (tim) expression are initiated at the first instar (L1) larval stage or during metamorphosis, respectively. Because CLOCK-CYCLE (CLK-CYC) heterodimers initiate circadian oscillator function by activating per and tim transcription, a Clk-GFP transgene was used to mark when late pacemaker neurons begin to develop. We were surprised to see that CLK-GFP was already expressed in four of five clusters of late pacemaker neurons during the third instar (L3) larval stage. CLK-GFP is only detected in postmitotic neurons from L3 larvae, suggesting that these four late pacemaker neuron clusters are formed before the L3 larval stage. A GFP-cyc transgene was used to show that CYC, like CLK, is also expressed exclusively in pacemaker neurons from L3 larval brains, demonstrating that CLK-CYC is not sufficient to activate per and tim in late pacemaker neurons at the L3 larval stage. These results suggest that most late pacemaker neurons develop days before novel factors activate circadian oscillator function during metamorphosis.

Impaired cholesterol metabolism and enhanced atherosclerosis in clock mutant mice.

BACKGROUND: Clock is a key transcription factor that positively controls circadian regulation. However, its role in plasma cholesterol homeostasis and atherosclerosis has not been studied. METHODS AND RESULTS: We show for the first time that dominant-negative Clock mutant protein (Clock(Δ19/Δ19)) enhances plasma cholesterol and atherosclerosis in 3 different mouse models. Detailed analyses revealed that Clk(Δ19/Δ19)Apoe(-/-) mice display hypercholesterolemia resulting from the accumulation of apolipoprotein B48-containing cholesteryl ester-rich lipoproteins. Physiological studies showed that enhanced cholesterol absorption by the intestine contributes to hypercholesterolemia. Molecular studies indicated that the expression of Niemann Pick C1 Like 1, Acyl-CoA:Cholesterol acyltransferase 1, and microsomal triglyceride transfer protein in the intestines of Clk(Δ19/Δ19)Apoe(-/-) mice was high and that enterocytes assembled and secreted more chylomicrons. Furthermore, we identified macrophage dysfunction as another potential cause of increased atherosclerosis in Clk(Δ19/Δ19)Apoe(-/-) mice. Macrophages from Clk(Δ19/Δ19)Apoe(-/-) mice expressed higher levels of scavenger receptors and took up more modified lipoproteins compared with Apoe(-/-) mice, but they expressed low levels of ATP binding casette protein family A member 1 and were defective in cholesterol efflux. Molecular studies revealed that Clock regulates ATP binding casette protein family A member 1 expression in macrophages by modulating upstream transcription factor 2 expression. CONCLUSIONS: Clock(Δ19/Δ19) protein enhances atherosclerosis by increasing intestinal cholesterol absorption, augmenting uptake of modified lipoproteins by macrophages, and reducing cholesterol efflux from macrophages. These studies establish that circadian Clock activity is crucial in maintaining low plasma cholesterol levels and in reducing atherogenesis in mice.

Quantification of interactions among circadian clock proteins via surface plasmon resonance.

Circadian clock is an internal time keeping system recurring 24 h daily rhythm in physiology and behavior of organisms. Circadian clock contains transcription and translation feedback loop involving CLOCK/NPAS2, BMAL1, Cry1/2, and Per1/2. In common, heterodimer of CLOCK/NPAS2 and BMAL1 binds to EBOX element in the promoter of Per and Cry genes in order to activate their transcription. CRY and PER making heterodimeric complexes enter the nucleus in order to inhibit their own BMAL1-CLOCK-activated transcription. The aim of this study was to investigate and quantify real-time binding affinities of clock proteins among each other on and off DNA modes using surface plasmon resonance. The pairwise interaction coefficients among clock proteins, as well as interaction of PER2, CRY2, and PER2 : CRY2 proteins with BMAL1 : CLOCK complex in the presence and absence of EBOX motif have been investigated via analysis of surface plasmon resonance data with pseudo first-order reaction kinetics approximation and via nonlinear regression curve fitting. The results indicated that CRY2 and PER2, BMAL1, and CLOCK proteins form complexes in vitro and that PER2, CRY2 and PER2 : CRY2 complex have similar affinities toward BMAL1 : CLOCK complex. CRY2 protein had the highest affinity toward EBOX complex, whereas PER2 and CRY2 : PER2 complexes displayed low affinity toward EBOX complex. The quantification of the interaction between clock proteins is critical to understand the operation mechanism of the biological clock and to address the behavioral and physiological disorders, and it will be useful for the design of new drugs toward clock-related diseases.

The Essential and the Nonessential Roles of Four Clock Elements in the Circadian Rhythm of Metarhiziumrobertsii.

FRQ (frequency protein), FRH (FRQ-interacting RNA helicase), and WC1 and WC2 (white collar proteins) are major clock elements that govern the circadian rhythm in Neurosporacrassa. However, deletion of frh is lethal for the viability of N. crassa, making it elusive whether FRH is essential or nonessential for the circadian rhythm. This needs clarification in a fungus where frh deletion is not lethal. Here, the nuclear FRH ortholog proved nonessential for the circadian rhythm of Metarhiziumrobertsii. The nucleocytoplasmic shuttling of M. robertsii FRQ, WC1, and WC2 orthologs was light-dependent. Yeast two-hybrid assay validated interactions of FRQ with FRH and WC1 instead of FRH with WC1 and WC2 or FRQ with WC2. The circadian rhythm well, shown as conidiation rings of tint and dark in 15 d-old plate cultures grown at 25 °C in a light/dark cycle of 12:12, was abolished in the absence of frq or wc1, partially disturbed in the absence of wc2, but unaffected in the absence of frh. These results indicate a requirement of either FRQ or WC1 instead of FRH for the fungal circadian rhythm. Further analyses of frq and frh mutants revealed the dispensable and the limited roles of FRQ and FRH in the insect-pathogenic lifecycle of M. robertsii, respectively.

Impact of Circadian Rhythms on the Development and Clinical Management of Genitourinary Cancers.

The circadian system is an innate clock mechanism that governs biological processes on a near 24-hour cycle. Circadian rhythm disruption (i.e., misalignment of circadian rhythms), which results from the lack of synchrony between the master circadian clock located in the suprachiasmatic nuclei (SCN) and the environment (i.e., exposure to day light) or the master clock and the peripheral clocks, has been associated with increased risk of and unfavorable cancer outcomes. Growing evidence supports the link between circadian disruption and increased prevalence and mortality of genitourinary cancers (GU) including prostate, bladder, and renal cancer. The circadian system also plays an essential role on the timely implementation of chronopharmacological treatments, such as melatonin and chronotherapy, to reduce tumor progression, improve therapeutic response and reduce negative therapy side effects. The potential benefits of the manipulating circadian rhythms in the clinical setting of GU cancer detection and treatment remain to be exploited. In this review, we discuss the current evidence on the influence of circadian rhythms on (disease) cancer development and hope to elucidate the unmet clinical need of defining the extensive involvement of the circadian system in predicting risk for GU cancer development and alleviating the burden of implementing anti-cancer therapies.

Characterizing the relative abundance of circadian transcription factors in diapausing and nondiapausing Northern house mosquitoes.

The Northern house mosquito (Culex pipiens) is a major vector of West Nile virus. To survive harsh conditions in winter adult females of Cx. pipiens enter a state of arrested reproductive development called diapause. Diapause is triggered by the short daylengths of late summer and early fall. The methods by which Cx. pipiens measures daylength are still unknown. However, it is suspected that clock genes, which provide information on daylength, may also regulate diapause. The proteins produced by these genes often cycle in abundance throughout the day in diapausing and nondiapausing insects. Two clock genes suspected to control diapause are cycle (cyc) and Par domain protein1 (Pdp1) as they encode circadian transcription factors that may regulate genes that are involved in diapause. Using Western blotting we measured the relative protein abundance of CYC and PDP1 throughout the day in the whole bodies and the heads of Cx. pipiens reared under either long-day, diapause-averting conditions or short-day, diapause-inducing conditions. We found that in whole bodies there was no significant oscillation of CYC or PDP1 abundance in both long day and short day-reared mosquitoes. In the heads of long day-reared mosquitoes both CYC and PDP1 cycled. In contrast, only PDP1 abundance showed diel differences in abundance in the heads of short day-reared mosquitoes. These data bring us one step closer to understanding the role that CYC and PDP1 may play in regulating diapause and other biological processes.

The role of spatiotemporal organization and dynamics of clock complexes in circadian regulation.

Circadian clocks are cell autonomous timekeepers that regulate ∼24-h oscillations in the expression of many genes and control rhythms in nearly all our behavior and physiology. Almost every cell in the human body has a molecular clock and networks of cells containing clock proteins orchestrate daily rhythms in many physiological processes, from sleep-wake cycles to metabolism to immunity. All eukaryotic circadian clocks are based on transcription-translation delayed negative feedback loops in which activation of core clock genes is negatively regulated by their cognate protein products. Our current understanding of circadian clocks has been accumulated from decades of genetic and biochemical experiments, however, what remains poorly understood is how clock proteins, genes, and mRNAs are spatiotemporally organized within live clock cells and how such subcellular organization affects circadian rhythms at the single cell level. Here, we review recent progress in understanding how clock proteins and genes are spatially organized within clock cells over the circadian cycle and the role of such organization in generating circadian rhythms and highlight open questions for future studies.

Co-Expression Network Analysis of Micro-RNAs and Proteins in the Alzheimer's Brain: A Systematic Review of Studies in the Last 10 Years.

MicroRNAs (miRNAs) are small non-coding nucleic acids that can regulate post-transcriptional gene expression by binding to complementary sequences of target mRNA. Evidence showed that dysregulated miRNA expression may be associated with neurological conditions such as Alzheimer's disease (AD). In this study, we combined the results of two independent systematic reviews aiming to unveil the co-expression network of miRNAs and proteins in brain tissues of AD patients. Twenty-eight studies including a total of 113 differentially expressed miRNAs (53 of them validated by qRT-PCR), and 26 studies including a total of 196 proteins differentially expressed in AD brains compared to healthy age matched controls were selected. Pathways analyses were performed on the results of the two reviews and 39 common pathways were identified. A further bioinformatic analysis was performed to match miRNA and protein targets with an inverse relation. This revealed 249 inverse relationships in 28 common pathways, representing new potential targets for therapeutic intervention. A meta-analysis, whenever possible, revealed miR-132-3p and miR-16 as consistently downregulated in late-stage AD across the literature. While no inverse relationships between miR-132-3p and proteins were found, miR-16's inverse relationship with CLOCK proteins in the circadian rhythm pathway is discussed and therapeutic targets are proposed. The most significant miRNA dysregulated pathway highlighted in this review was the hippo signaling pathway with p = 1.66 × 10-9. Our study has revealed new mechanisms for AD pathogenesis and this is discussed along with opportunities to develop novel miRNA-based drugs to target these pathways.

Circadian Phosphorylation of CLOCK and BMAL1.

Daily rhythms of behaviors and physiologies are driven by transcriptional-translational negative feedback loops of clock genes and encoded clock proteins (Bass and Takahashi Science 330:1349-1354, 2010; Brown et al. Dev Cell 22:477-487, 2012). Posttranslational modifications of clock proteins, including protein phosphorylation, play an essential role for normal oscillation of the circadian clock through regulation of their activities, stabilities, interactions, and intracellular localization (Gallego and Virshup Nat Rev Mol Cell Biol 8:139-148, 2007; Hirano et al. Nat Struct Mol Biol 23:1053-1060, 2016). In this chapter, we describe detailed methods for quantitative analysis of phosphorylation levels of clock proteins, particularly focusing on circadian phosphorylation of CLOCK, BMAL1, and their complex (Yoshitane et al. Mol Cell Biol 29:3675-3686, 2009).

The circadian machinery links metabolic disorders and depression: A review of pathways, proteins and potential pharmacological interventions.

Circadian rhythms are responsible for regulating a number of physiological processes. The central oscillator is located within the suprachiasmatic nucleus (SCN) of the hypothalamus and the SCN synchronises the circadian clocks that are found in our peripheral organs through neural and humoral signalling. At the molecular level, biological clocks consist of transcription-translation feedback loops (TTFLs) and these pathways are influenced by transcription factors, post-translational modifications, signalling pathways and epigenetic modifiers. When disruptions occur in the circadian machinery, the activities of the proteins implicated in this network and the expression of core clock or clock-controlled genes (CCGs) can be altered. Circadian misalignment can also arise when there is desychronisation between our internal clocks and environmental stimuli. There is evidence in the literature demonstrating that disturbances in the circadian rhythm contribute to the pathophysiology of several diseases and disorders. This includes the metabolic syndrome and recently, it has been suggested that the 'circadian syndrome' may be a more appropriate term to use to not only describe the cardio-metabolic risk factors but also the associated comorbidities. Here we overview the molecular architecture of circadian clocks in mammals and provide insight into the effects of shift work, exposure to artificial light, food intake and stress on the circadian rhythm. The relationship between circadian rhythms, metabolic disorders and depression is reviewed and this is a topic that requires further investigation. We also describe how particular proteins involved in the TTFLs can be potentially modulated by small molecules, including pharmacological interventions and dietary compounds.

Small-molecule modulators of the circadian clock: Pharmacological potentials in circadian-related diseases.

Disruption of circadian oscillations has a wide-ranging impact on health, with the potential to induce the development of clock-related diseases. Small-molecule modulators of the circadian clock (SMMCC) target core or noncore clock proteins, modulating physiological effects as a consequence of agonist, inverse agonist, or antagonist interference. These pharmacological modulators are usually identified using chemical screening of large libraries of active compounds. However, target-based screens, chemical optimization, and circadian crystallography have recently assisted in the identification of these compounds. In this review, we focus on established and novel SMMCCs targeting both core and noncore clock proteins, identifying their circadian targets, detailed circadian effects, and specific physiological effects. In addition, we discuss their therapeutic potential for the treatment of diverse clock-related disorders (such as metabolic-associated disorders, autoimmune diseases, mood disorders, and cancer) and as chronotherapeutics. Future perspectives are also considered, such as clinical trials, and potential safety hazards, including those in the absence of clinical trials.

Pharmacological Interventions to Circadian Clocks and Their Molecular Bases.

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.

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

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-1ß, 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.

Corrigendum: Low-Light Dependence of the Magnetic Field Effect on Cryptochromes: Possible Relevance to Plant Ecology.

[This corrects the article DOI: 10.3389/fpls.2018.00121.].