Deubiquitinating enzyme USP9X regulates cellular clock function by modulating the ubiquitination and degradation of a core circadian protein BMAL1

2018 ◽  
Vol 475 (8) ◽  
pp. 1507-1522 ◽  
Author(s):  
Yang Zhang ◽  
Chunyan Duan ◽  
Jing Yang ◽  
Suping Chen ◽  
Qing Liu ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Circadian Clock ◽  
Clock Genes ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Regulatory Function ◽  
Spectrometry Analysis ◽  
The Core ◽  
Clock Proteins ◽  
Clock Protein

Living organisms on the earth maintain a roughly 24 h circadian rhythm, which is regulated by circadian clock genes and their protein products. Post-translational modifications of core clock proteins could affect the circadian behavior. Although ubiquitination of core clock proteins was studied extensively, the reverse process, deubiquitination, has only begun to unfold and the role of this regulation on circadian function is not completely understood. Here, we use affinity purification and mass spectrometry analysis to identify probable ubiquitin carboxyl-terminal hydrolase FAF-X (USP9X) as an interacting protein of the core clock protein aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL or BMAL1). Through biochemical experiments, we discover that USP9X reduces BMAL1 ubiquitination, enhances its stability, and increases its protein level, leading to the elevated transcriptional activity. Bioluminescence measurement reveals that USP9X knockdown decreases the amplitude of the cellular circadian rhythm but the period and phase are not affected. Our experiments find a new regulator for circadian clock at the post-translational level and demonstrate a different regulatory function for the circadian clock through the deubiquitination and the up-regulation of the core clock protein BMAL1 in the positive limb of the transcription–translation feedback loop.

scholarly journals Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms

2020 ◽  
Author(s):  
Yangbo Xiao ◽  
Ye Yuan ◽  
Mariana Jimenez ◽  
Neeraj Soni ◽  
Swathi Yadlapalli
Keyword(s):  
Gene Expression ◽  
Circadian Rhythms ◽  
Nuclear Envelope ◽  
Circadian Clock ◽  
Clock Genes ◽  
Circadian Clocks ◽  
Spatiotemporal Organization ◽  
Clock Proteins ◽  
Expression Behavior ◽  
Clock Protein

ABSTRACTCircadian clocks regulate ∼24 hour oscillations in gene expression, behavior, and physiology. While the molecular and neural mechanisms of circadian rhythms are well characterized, how cellular organization of clock components controls circadian clock regulation remains poorly understood. Here, we elucidate how clock proteins regulate circadian rhythms by controlling the spatiotemporal organization of clock genes. Using high-resolution live imaging techniques we demonstrate that Drosophila clock proteins are concentrated in a few discrete foci and are organized at the nuclear envelope; these results are in contrast to longstanding expectations that clock proteins are diffusely distributed in the nucleus. We also show that clock protein foci are highly dynamic and change in number, size, and localization over the circadian cycle. Further, we demonstrate that clock genes are positioned at the nuclear periphery by the clock proteins precisely during the circadian repression phase, suggesting that subnuclear localization of clock genes plays an important role in the control of rhythmic gene expression. Finally, we show that Lamin B receptor, a nuclear envelope protein, is required for peripheral localization of clock protein foci and clock genes and for normal circadian rhythms. These results reveal that clock proteins form dynamic nuclear foci and play a hitherto unexpected role in the subnuclear reorganization of clock genes to control circadian rhythms, identifying a novel mechanism of circadian regulation. Our results further suggest a new role for clock protein foci in the clustering of clock-regulated genes during the repression phase to control gene co-regulation and circadian rhythms.SIGNIFICANCEAlmost all living organisms have evolved circadian clocks to tell time. Circadian clocks regulate ∼24-hour oscillations in gene expression, behavior and physiology. Here, we reveal the surprisingly sophisticated spatiotemporal organization of clock proteins and clock genes and its critical role in circadian clock function. We show, in contrast to current expectations, that clock proteins are concentrated in a few discrete, dynamic nuclear foci at the nuclear envelope during the repression phase. Further, we uncovered several unexpected features of clock protein foci, including their role in positioning the clock genes at the nuclear envelope precisely during the repression phase to enable circadian rhythms. These studies provide fundamental new insights into the cellular mechanisms of circadian rhythms and establish direct links between nuclear organization and circadian clocks.

scholarly journals Study of the modulatory mechanism of the miR-182-Clock axis in circadian rhythm disturbance after hypoxic–ischemic brain damage

2020 ◽  
Vol 18 ◽  
pp. 205873922092915
Author(s):  
Dezhan Li ◽  
Wei Huang ◽  
Fang Yang ◽  
Bin Li ◽  
Shanshan Cai
Keyword(s):  
Circadian Rhythm ◽  
Pineal Gland ◽  
Circadian Clock ◽  
Sleep Disorders ◽  
Clock Genes ◽  
Ischemic Brain ◽  
The Core ◽  
Circadian Rhythm Disorder ◽  
Pineal Function ◽  
Rhythm Disorder

Hypoxic–ischemic encephalopathy (HIE) in neonates can lead to severe chronic neurological deficit, including mental retardation, epilepsy, and sleep–wake cycle (SWC) disorder. Among these defects, little is known about the molecular mechanism of circadian rhythm disorder after HIE. Therefore, further study of sleep problems and its mechanism in HIE children will provide new ideas for clinical treatment of HIE children. For pediatric patients with cerebral ischemia, somnipathy often occurs due to visual and airway abnormalities. From May 2010 to August 2013, 128 newborns with history of HIE were followed up. Meanwhile, 88 normal full-term newborns in the same period were taken as the control group. The clinical data of the patients were collected and the sleep status was assessed by questionnaire. To establish the hypoxic–ischemic brain injury model of neonatal rats and analyze the mechanism of mir-182 in the circadian rhythm disorder caused by pineal function injury. The core clock genes during the regulation of the circadian clock were explored by bioinformatics methods. Patients’ sleep quality was affected by the circadian rhythm and respiratory problems; the pineal gland can regulate the core clock genes in the circadian clock during regulation. miR-182 was highly expressed in the pineal gland after hypoxic–ischemic brain damage (HIBD). Children with mild and moderate HIE showed significant sleep disorders in varying degrees, which provided a clinical basis for improving the long-term prognosis of children with HIE through targeted treatment of sleep disorders. MiR-182 is highly expressed in the pineal gland and is related to the expression of CLOCK protein. CLOCK gene is the target gene of miR-182, which provides a new target for the treatment of rhythm disorder related to the damage of pineal function caused by HIBD.

scholarly journals Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

2021 ◽  
pp. 074873042199994
Author(s):  
Rosa Eskandari ◽  
Lalanthi Ratnayake ◽  
Patricia L. Lakin-Thomas
Keyword(s):  
Circadian Rhythms ◽  
Clock Genes ◽  
Nutrient Sensing ◽  
Mass Spectrometry Analysis ◽  
Growth Responses ◽  
Tor Pathway ◽  
Spectrometry Analysis ◽  
Binding Partner ◽  
Binding Partners ◽  
Free Running

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes ( frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation ( vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ gtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ gtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ gtr2 is defective in entrainment. In all of these assays, Δ gtr2 is similar to Δ vta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

scholarly journals Circadian Rhythm: Potential Therapeutic Target for Atherosclerosis and Thrombosis

2021 ◽  
Vol 22 (2) ◽  
pp. 676
Author(s):  
Andy W. C. Man ◽  
Huige Li ◽  
Ning Xia
Keyword(s):  
Circadian Rhythm ◽  
Cardiovascular Diseases ◽  
Circadian Rhythms ◽  
Circadian Clock ◽  
Therapeutic Target ◽  
Environmental Changes ◽  
Clock Genes ◽  
Vascular System ◽  
Peripheral Tissues ◽  
Inflammatory Processes

Every organism has an intrinsic biological rhythm that orchestrates biological processes in adjusting to daily environmental changes. Circadian rhythms are maintained by networks of molecular clocks throughout the core and peripheral tissues, including immune cells, blood vessels, and perivascular adipose tissues. Recent findings have suggested strong correlations between the circadian clock and cardiovascular diseases. Desynchronization between the circadian rhythm and body metabolism contributes to the development of cardiovascular diseases including arteriosclerosis and thrombosis. Circadian rhythms are involved in controlling inflammatory processes and metabolisms, which can influence the pathology of arteriosclerosis and thrombosis. Circadian clock genes are critical in maintaining the robust relationship between diurnal variation and the cardiovascular system. The circadian machinery in the vascular system may be a novel therapeutic target for the prevention and treatment of cardiovascular diseases. The research on circadian rhythms in cardiovascular diseases is still progressing. In this review, we briefly summarize recent studies on circadian rhythms and cardiovascular homeostasis, focusing on the circadian control of inflammatory processes and metabolisms. Based on the recent findings, we discuss the potential target molecules for future therapeutic strategies against cardiovascular diseases by targeting the circadian clock.

scholarly journals Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter

2020 ◽  
Author(s):  
Bo Wei ◽  
Patrick Willems ◽  
Jingjing Huang ◽  
Caiping Tian ◽  
Jing Yang ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Technological Advancement ◽  
Amino Acid Residues ◽  
Cysteine Oxidation ◽  
Spectrometry Analysis ◽  
Mixed Disulfide ◽  
And Function

ABSTRACTIn proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1–like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues remained enigmatic. Here, we present a technological advancement to identify in situ sulfenylated cysteines directly by means of the transgenic Yap1 probe. In Arabidopsis thaliana cells, after an initial affinity purification and a tryptic digestion, we further enriched the mixed disulfide-linked peptides with an antibody targeting the YAP1C-derived peptide (C598SEIWDR) that entails the redox-active cysteine. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

scholarly journals MECHANISMS IN ENDOCRINOLOGY: A sense of time of the glucocorticoid circadian clock: from the ontogeny to the diagnosis of Cushing’s syndrome

2018 ◽  
Vol 179 (1) ◽  
pp. R1-R18 ◽  
Author(s):  
Ayrton Custodio Moreira ◽  
Sonir Rauber Antonini ◽  
Margaret de Castro
Keyword(s):  
Circadian Rhythm ◽  
Circadian Clock ◽  
Clock Genes ◽  
Circadian Variation ◽  
Circadian System ◽  
Feedback Loops ◽  
Adrenal Axis ◽  
Sense Of Time ◽  
Pituitary Adrenal Axis ◽  
Hierarchical Manner

The circadian rhythm of glucocorticoids has long been recognised within the last 75 years. Since the beginning, researchers have sought to identify basic mechanisms underlying the origin and emergence of the corticosteroid circadian rhythmicity among mammals. Accordingly, Young, Hall and Rosbash, laureates of the 2017 Nobel Prize in Physiology or Medicine, as well as Takahashi’s group among others, have characterised the molecular cogwheels of the circadian system, describing interlocking transcription/translation feedback loops essential for normal circadian rhythms. Plasma glucocorticoid circadian variation depends on the expression of intrinsic clock genes within the anatomic components of the hypothalamic–pituitary–adrenal axis, which are organised in a hierarchical manner. This review presents a general overview of the glucocorticoid circadian clock mechanisms, highlighting the ontogeny of the pituitary–adrenal axis diurnal rhythmicity as well as the involvement of circadian rhythm abnormalities in the physiopathology and diagnosis of Cushing’s disease.

scholarly journals Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti

2019 ◽  
Vol 116 (38) ◽  
pp. 19136-19144 ◽  
Author(s):  
Giel P. Göertz ◽  
Joyce W. M. van Bree ◽  
Anwar Hiralal ◽  
Bas M. Fernhout ◽  
Carmen Steffens ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Zika Virus ◽  
Affinity Purification ◽  
Mechanistic Model ◽  
Virus Transmission ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Small Interfering Rnas ◽  
Spectrometry Analysis ◽  
Mosquito Infection

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

scholarly journals “Gut Microbiota-Circadian Clock Axis” in Deciphering the Mechanism Linking Early-Life Nutritional Environment and Abnormal Glucose Metabolism

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Liyuan Zhou ◽  
Lin Kang ◽  
Xinhua Xiao ◽  
Lijing Jia ◽  
Qian Zhang ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Circadian Rhythm ◽  
Gut Microbiota ◽  
Circadian Clock ◽  
Early Life ◽  
Metabolic Diseases ◽  
Clock Genes ◽  
Early Stage ◽  
Adult Life ◽  
Metabolic Memory

The prevalence of diabetes mellitus (DM) has been increasing dramatically worldwide, but the pathogenesis is still unknown. A growing amount of evidence suggests that an abnormal developmental environment in early life increases the risk of developing metabolic diseases in adult life, which is referred to as the “metabolic memory” and the Developmental Origins of Health and Disease (DOHaD) hypothesis. The mechanism of “metabolic memory” has become a hot topic in the field of DM worldwide and could be a key to understanding the pathogenesis of DM. In recent years, several large cohort studies have shown that shift workers have a higher risk of developing type 2 diabetes mellitus (T2DM) and worse control of blood glucose levels. Furthermore, a maternal high-fat diet could lead to metabolic disorders and abnormal expression of clock genes and clock-controlled genes in offspring. Thus, disorders of circadian rhythm might play a pivotal role in glucose metabolic disturbances, especially in terms of early adverse nutritional environments and the development of metabolic diseases in later life. In addition, as a peripheral clock, the gut microbiota has its own circadian rhythm that fluctuates with periodic feeding and has been widely recognized for its significant role in metabolism. In light of the important roles of the gut microbiota and circadian clock in metabolic health and their interconnected regulatory relationship, we propose that the “gut microbiota-circadian clock axis” might be a novel and crucial mechanism to decipher “metabolic memory.” The “gut microbiota-circadian clock axis” is expected to facilitate the future development of a novel target for the prevention and intervention of diabetes during the early stage of life.

CORTISOL CIRCADIAN RHYTHM AND INSULIN RESISTANCE IN MUSCLE: EFFECT OF DOSING AND TIMING OF HYDROCORTISONE EXPOSURE ON INSULIN SENSITIVITY IN SYNCHRONIZED MUSCLE CELLS.

2020 ◽  
Author(s):  
Mariarosaria Negri ◽  
Claudia Pivonello ◽  
Chiara Simeoli ◽  
Gilda Di Gennaro ◽  
Mary Anna Venneri ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Circadian Rhythm ◽  
Insulin Sensitivity ◽  
Circadian Clock ◽  
Insulin Signaling ◽  
Clock Genes ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Circadian Expression

Introduction/Aim: Circadian rhythm disruption is emerging as a risk factor for metabolic disorders and particularly, alterations in clock genes circadian expression have been shown to influence insulin sensitivity. Recently, the reciprocal interplay between the circadian clock machinery and HPA axis has been largely demonstrated: the circadian clock may control the physiological circadian endogenous glucocorticoids secretion and action; glucocorticoids, in turn, are potent regulator of the circadian clock and their inappropriate replacement has been associated with metabolic impairment. The aim of the current study was to investigate in vitro the interaction between the timing-of-the-day exposure to different hydrocortisone (HC) concentrations on muscle insulin sensitivity. Methods: Serum-shock synchronized mouse skeletal muscle C2C12 cells were exposed to different HC concentrations recapitulating the circulating daily physiological cortisol profile (standard cortisol profile), the circulating daily cortisol profile that reached in adrenal insufficient (AI) patients treated with once-daily MR-HC (flat cortisol profile) and treated with thrice-daily of conventional IR-HC (steep cortisol profile). The 24 hrs spontaneous oscillation of the clock genes in synchronized C2C12 cells was used to align the timing for in vitro HC exposure (Bmal1 acrophase, midphase and bathyphase) with the reference times of cortisol peaks in AI treated with IR-HC (8 am, 1 pm, 6 pm). A panel of 84 insulin sensitivity related genes and intracellular insulin signaling proteins were analyzed by RT-qPCR and western blot, respectively. Results: Only the steep profile, characterized by a higher HC exposure during Bmal1 bathyphase, produced significant downregulation in 21 insulin sensitivity-related genes. Among these, Insr, Irs1, Irs2, Pi3kca and Adipor2 were downregulated when compared the flat to the standard or steep profile. Reduced intracellular IRS1 Tyr608, AKT Ser473, AMPK Thr172 and ACC Ser79 phosphorylations were also observed. Conclusions: The current study demonstrated that is late-in-the-day cortisol exposure that modulates insulin sensitivity-related genes expression and intracellular insulin signaling in skeletal muscle cells.

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