Elucidation of Master Allostery Essential for Circadian Clock Oscillation in Cyanobacteria

Spatio-temporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor–transfer events at Ser431 and Thr432. The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser431-dependent phospho-switch in the C-terminal domain of KaiC and ADP release from its N-terminal domain during ATPase cycle. An engineered KaiC–protein oscillator consisting of a minimal set of the identified master allosteric events exhibited mono-phosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple post-translational biochemical circadian oscillators.One Sentence SummaryCoupling between a phospho-switch and KaiC ATPase-dependent nucleotide exchange drives the cyanobacterial circadian clock.

Overlap of expression and alignment of diurnal and circadian rhythmicity in the human blood transcriptome with organ and tissue specific rhythmicity in a non-human primate

BACKGROUND: Twenty-four-hour rhythmicity in transcriptomes of tissues and organs is driven by local circadian oscillators, systemic factors, the central circadian pacemaker, and light-dark cycles. This rhythmicity is to some extent organ- and tissue-specific such that the sets of rhythmic transcripts or their timing are different across tissues/organs. Monitoring rhythmicity of tissues and organs holds promise for circadian medicine, but in humans most tissues and organs are not easily accessible. To investigate the extent to which rhythmicity in the human blood transcriptome reflects rhythmicity in tissues and organs, we compared the overlap and timing of rhythmic transcripts in human blood and rhythmic transcripts in 64 tissues/organs of the baboon. METHODS: Rhythmicity in the transcriptomes of humans and baboons were compared using set logic, circular cross-correlation, circular clustering, functional enrichment analyses and partial least squares regression. RESULTS: Of the 759 orthologous genes that were rhythmic in human blood, 652 (86%) were also rhythmic in at least one baboon tissue. Most of these genes were associated with basic processes such as transcription and protein homeostasis. 109 (17%) of the 652 overlapping rhythmic genes were reported as rhythmic in only one baboon tissue or organ and several of these genes have tissue/organ-specific functions. Analysis of the alignment between baboon and human transcriptomes showed that in these diurnal species, rhythmicity is aligned with the onset, rather than midpoint or end of light period. In both species, the timing of rhythmic transcripts displayed prominent "night" and "day" clusters, with genes in the dark cluster associated with translation. The timing of human and baboon transcriptomes was significantly correlated in 25 tissue/organs with an average earlier timing of 3.21 h (SD 2.47 h) in human blood. CONCLUSIONS: The human blood transcriptome contains sets of rhythmic genes that overlap with rhythmic genes of tissues/organs, some of which are tissue/organ-specific, in the baboon. The rhythmic sets vary across tissues/organs but the timing of most rhythmic genes is similar across human blood and baboon tissues/organs. These results have implications for our understanding of the regulation of rhythmicity across tissues/organs and species and development of blood transcriptome-based biomarkers for rhythmicity in tissues and organs.

Intercellular coupling between peripheral circadian oscillators by TGF-β signaling

Coupling between cell-autonomous circadian oscillators is crucial to prevent desynchronization of cellular networks and disruption of circadian tissue functions. While neuronal oscillators within the mammalian central clock, the suprachiasmatic nucleus, couple intercellularly, coupling among peripheral oscillators is controversial and the molecular mechanisms are unknown. Using two- and three-dimensional mammalian culture models in vitro (mainly human U-2 OS cells) and ex vivo, we show that peripheral oscillators couple via paracrine pathways. We identify transforming growth factor–β (TGF-β) as peripheral coupling factor that mediates paracrine phase adjustment of molecular clocks through transcriptional regulation of core-clock genes. Disruption of TGF-β signaling causes desynchronization of oscillator networks resulting in reduced amplitude and increased sensitivity toward external zeitgebers. Our findings reveal an unknown mechanism for peripheral clock synchrony with implications for rhythmic organ functions and circadian health.

A Global Search for novel Transcription Factors impacting the Neurospora crassa Circadian Clock

Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and WC-2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 transcription factors that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated transcription factors, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.

Divergent Importance of Chronobiological Considerations in High- and Low-dose Melatonin Therapies

Melatonin has been used preclinically and clinically for different purposes. Some applications are related to readjustment of circadian oscillators, others use doses that exceed the saturation of melatonin receptors MT1 and MT2 and are unsuitable for chronobiological purposes. Conditions are outlined for appropriately applying melatonin as a chronobiotic or for protective actions at elevated levels. Circadian readjustments require doses in the lower mg range, according to receptor affinities. However, this needs consideration of the phase response curve, which contains a silent zone, a delay part, a transition point and an advance part. Notably, the dim light melatonin onset (DLMO) is found in the silent zone. In this specific phase, melatonin can induce sleep onset, but does not shift the circadian master clock. Although sleep onset is also under circadian control, sleep and circadian susceptibility are dissociated at this point. Other limits of soporific effects concern dose, duration of action and poor individual responses. The use of high melatonin doses, up to several hundred mg, for purposes of antioxidative and anti-inflammatory protection, especially in sepsis and viral diseases, have to be seen in the context of melatonin’s tissue levels, its formation in mitochondria, and detoxification of free radicals.

Thymus-Pineal Gland Axis: Revisiting Its Role in Human Life and Ageing

For years the thymus gland (TG) and the pineal gland (PG) have been subject of increasingly in-depth studies, but only recently a link that can associate the activities of the two organs has been identified. Considering, on the one hand, the well-known immune activity of thymus and, on the other, the increasingly emerging immunological roles of circadian oscillators and the rhythmically secreted main pineal product, melatonin, many studies aimed to analyse the possible existence of an interaction between these two systems. Moreover, data confirmed that the immune system is functionally associated with the nervous and endocrine systems determining an integrated dynamic network. In addition, recent researches showed a similar, characteristic involution process both in TG and PG. Since the second half of the 20th century, evidence led to the definition of an effectively interacting thymus-pineal axis (TG-PG axis), but much has to be done. In this sense, the aim of this review is to summarize what is actually known about this topic, focusing on the impact of the TG-PG axis on human life and ageing. We would like to give more emphasis to the implications of this dynamical interaction in a possible therapeutic strategy for human health. Moreover, we focused on all the products of TG and PG in order to collect what is known about the role of peptides other than melatonin. The results available today are often unclear and not linear. These peptides have not been well studied and defined over the years. In this review we hope to awake the interest of the scientific community in them and in their future pharmacological applications.

Bile acid metabolism and circadian rhythms

Circadian rhythms are biological systems that synchronize cellular circadian oscillators and regulate nutrient absorption and utilization. Bile acids are important modulators that facilitate nutrient absorption and regulate energy metabolism. Bile acid metabolism and circadian rhythms are related to metabolic diseases, and their intersections have not been summarized clearly up to now. This review summarizes the molecular association between circadian rhythms and bile acid metabolism and points out future perspectives and potential therapeutic targets in metabolic diseases.

Housing temperature affects the circadian rhythm of hepatic metabolism and clock genes

Environmental temperature remarkably impacts on metabolic homeostasis, raising a serious concern about the optimum housing temperature for translational studies. Recent studies suggested that mice should be housed slightly below their thermoneutral temperature (26°C). On the other hand, the external temperature, also known as a zeitgeber, can reset the circadian rhythm. However, whether housing temperature affects the circadian oscillators of the liver remains unknown. Therefore, we have compared the effect of two housing temperatures, namely 21°C (conventional; TC) and 26°C (thermoneutral; TN), on the circadian rhythms in mice. We found that the rhythmicity of food intake showed an advanced phase at TC, while the activity was more robust at TN, with a prolonged period onset. The serum levels of norepinephrine were remarkably induced at TC, but failed to oscillate rhythmically at both temperatures. Likewise, circulating glucose levels were increased but were non-rhythmic under TC. Both total cholesterol and triglycerides levels were induced at TN, but showed an advanced phase under TC. Additionally, the expression of hepatic metabolic genes and clock genes remained rhythmic at both temperatures, with the exception of G6Pase, Fasn, Cpt1a and Cry2, at TN. Nevertheless, the liver histology examination did not show any significant changes in response to housing temperature. Although the non-consistent trends of phase changes in each temperature, our results suggest a non-reductant role of temperature in mouse internal rhythmicity resetting. Thus, the temperature-controlled internal circadian synchronization within organs should be taken into consideration when optimizing housing temperature for mice.