Interaction of circadian clock proteins PER2 and CRY with BMAL1 and CLOCK

Background: Circadian oscillation of clock-controlled gene expression is mainly regulated at the transcriptional level. Heterodimers of CLOCK and BMAL1 act as activators of target gene transcription; however, interactions of PER and CRY proteins with the heterodimer abolish its transcriptional activation capacity. PER and CRY are therefore referred to as negative regulators of the circadian clock. To further elucidate the mechanism how positive and negative components of the clock interplay, we characterized the interactions of PER2, CRY1 and CRY2 with BMAL1 and CLOCK using a mammalian two-hybrid system and co-immunoprecipitation assays. Results: Both PER2 and the CRY proteins were found to interact with BMAL1 whereas only PER2 interacts with CLOCK. CRY proteins seem to have a higher affinity to BMAL1 than PER2. Moreover, we provide evidence that PER2, CRY1 and CRY2 bind to different domains in the BMAL1 protein. Conclusion: The regulators of clock-controlled transcription PER2, CRY1 and CRY2 differ in their capacity to interact with each single component of the BMAL1-CLOCK heterodimer and, in the case of BMAL1, also in their interaction sites. Our data supports the hypothesis that CRY proteins, especially CRY1, are stronger repressors than PER proteins

cGMP-Dependent Protein Kinase Type I Is Implicated in the Regulation of the Timing and Quality of Sleep and Wakefulness

Many effects of nitric oxide (NO) are mediated by the activation of guanylyl cyclases and subsequent production of the second messenger cyclic guanosine-3′,5′-monophosphate (cGMP). cGMP activates cGMP-dependent protein kinases (PRKGs), which can therefore be considered downstream effectors of NO signaling. Since NO is thought to be involved in the regulation of both sleep and circadian rhythms, we analyzed these two processes in mice deficient for cGMP-dependent protein kinase type I (PRKG1) in the brain. Prkg1 mutant mice showed a strikingly altered distribution of sleep and wakefulness over the 24 hours of a day as well as reductions in rapid-eye-movement sleep (REMS) duration and in non-REM sleep (NREMS) consolidation, and their ability to sustain waking episodes was compromised. Furthermore, they displayed a drastic decrease in electroencephalogram (EEG) power in the delta frequency range (1–4 Hz) under baseline conditions, which could be normalized after sleep deprivation. In line with the re-distribution of sleep and wakefulness, the analysis of wheel-running and drinking activity revealed more rest bouts during the activity phase and a higher percentage of daytime activity in mutant animals. No changes were observed in internal period length and phase-shifting properties of the circadian clock while chi-squared periodogram amplitude was significantly reduced, hinting at a less robust oscillator. These results indicate that PRKG1 might be involved in the stabilization and output strength of the circadian oscillator in mice. Moreover, PRKG1 deficiency results in an aberrant pattern, and consequently a reduced quality, of sleep and wakefulness, possibly due to a decreased wake-promoting output of the circadian system impinging upon sleep

Life time—circadian clocks, mitochondria and metabolism

A functional circadian clock has long been considered a selective advantage. Accumulating evidence shows that the clock coordinates a variety of physiological processes in order to schedule them to the optimal time of day and thus to synchronize metabolism to changes in external conditions. In mitochondria, both metabolic and cellular defense mechanisms are carefully regulated. Abnormal clock function, might influence mitochondrial function, resulting in decreased fitness of an organism

Differential gene expression between human schwannoma and control Schwann cells

The NF2 gene encodes the tumour suppressor protein merlin. The mutation of a single allele of this gene causes the autosomal dominantly inherited disease neurofibromatosis type 2 (NF2), which is characterized mainly by vestibular schwannoma carrying a second hit mutation. Complete lack of merlin is also found in spontaneous schwannomas and meningiomas. As the events leading to schwannoma development are largely unknown we investigated the differences in gene expression between schwannoma cells from NF2 patients and normal human primary Schwann cells by cDNA array analysis. We identified 41 genes whose expression levels differed by more than factor 2. Most of these clones were corroborated by real-time reverse transcription polymerase chain reaction analysis. By this method a total of seven genes with increased and seven genes with decreased mRNA levels in schwannoma compared with normal Schwann cells could be identified. Regulated clones, some of which not been described in Schwann cells earlier, included matrix metalloproteinase's, growth factors, growth factor receptors and tyrosine kinases

The mammalian circadian clock gene per2 modulates cell death in response to oxidative stress

Living in the earth's oxygenated environment forced organisms to develop strategies to cope with the damaging effects of molecular oxygen known as reactive oxygen species (ROS). Here, we show that Per2, a molecular component of the mammalian circadian clock, is involved in regulating a cell's response to oxidative stress. Mouse embryonic fibroblasts (MEFs) containing a mutation in the Per2 gene are more resistant to cytotoxic effects mediated by ROS than wild-type cells, which is paralleled by an altered regulation of bcl-2 expression in Per2 mutant MEFs. The elevated survival rate and alteration of NADH/NAD(+) ratio in the mutant cells is reversed by introduction of the wild-type Per2 gene. Interestingly, clock synchronized cells display a time dependent sensitivity to paraquat, a ROS inducing agent. Our observations indicate that the circadian clock is involved in regulating the fate of a cell to survive or to die in response to oxidative stress, which could have implications for cancer development and the aging process

Interaction of circadian clock proteins PER2 and CRY with BMAL1 and CLOCK-2

with or without addition of 0.3 μg . For each experiment (n = 3), values obtained for cells transfected with but without were set to 1. (B), (C) and (D) HER911 cells were co-transfected with , , and 0.001, 0.01, 0.1 or 1 μg (B) or 0.1 μg with and without addition of 0.3 μg (C, D). The amount of PER2-VP16, GAL4-BMAL1 and GFP was determined by Western blotting of total lysates (B, C) or nuclear and cytoplasmic fractions (D). Antibodies against CREB and HSP90 were used to verify correct cell fractionation. PER2-VP16 and GAL4-BMAL1 were normalized to GFP to correct for transfection efficiency. For both proteins, values obtained for cells transfected with , and only were set to 1 for each experiment (n = 4 for B and D, n = 5 for C). Blots are representative results from one experiment.<p><b>Copyright information:</b></p><p>Taken from "Interaction of circadian clock proteins PER2 and CRY with BMAL1 and CLOCK"</p><p>http://www.biomedcentral.com/1471-2199/9/41</p><p>BMC Molecular Biology 2008;9():41-41.</p><p>Published online 22 Apr 2008</p><p>PMCID:PMC2383916.</p><p></p

Interaction of circadian clock proteins PER2 and CRY with BMAL1 and CLOCK-7

Indicated expresssion plasmids. For each experiment (n = 6), values obtained for cells transfected with the luciferase reporter alone were set to 1. *** p < 0.001 as determined by student's t-test compared to all other columns. (B), (D) and (E) HER911 cells were co-transfected with and (B), (D) or (E). Total cell extracts (left panels) or immunoprecipitates using an anti-GFP antibody (right panels) were subjected to Western blotting using antibodies against PER2, CRY1 and CRY2, respectively. Blots are representative results from one experiment, all co-immunoprecipitations were repeated twice. WB Western Blot, IP immunoprecipitation.<p><b>Copyright information:</b></p><p>Taken from "Interaction of circadian clock proteins PER2 and CRY with BMAL1 and CLOCK"</p><p>http://www.biomedcentral.com/1471-2199/9/41</p><p>BMC Molecular Biology 2008;9():41-41.</p><p>Published online 22 Apr 2008</p><p>PMCID:PMC2383916.</p><p></p