Stoichiometric relationship among clock proteins determines robustness of circadian rhythms

The mammalian circadian oscillator is primarily driven by an essential negative feedback loop comprising a positive component, the CLOCK-BMAL1 complex, and a negative component, the PER-CRY complex. Numerous studies suggest that feedback inhibition of CLOCK-BMAL1 is mediated by time-dependent physical interaction with its direct target gene products PER and CRY, suggesting that the ratio between the negative and positive complexes must be important for the molecular oscillator and rhythm generation. We explored this idea by altering expression of clock components in fibroblasts derived from Per2(Luc) and Per mutant mice, a cell system extensively used to study in vivo clock mechanisms. Our data demonstrate that the stoichiometric relationship between clock components is critical for the robustness of circadian rhythms and provide insights into the mechanistic organization of the negative feedback loop. Our findings may explain why certain mutant mice or cells are arrhythmic, whereas others are rhythmic, and suggest that robustness of circadian rhythms can be increased even in wild-type cells by modulating the stoichiometry.     

Bifurcations in a mathematical model for circadian oscillations of clock genes

Circadian oscillations with a period of about 24h are observed in nearly all living organisms as conspicuous biological rhythms. In this paper, we investigate various kinds of bifurcation phenomena produced in a circadian oscillator model of Drosophila. In Drosophila, it is known that circadian oscillations in the levels of two proteins, PER and TIM, result from the negative feedback exerted by a PER-TIM complex on the expression of the per and tim genes that code for the two proteins. For studying circadian oscillations of proteins in Drosophila, a mathematical model has been proposed. The model cannot only account for regular circadian oscillations in environmental conditions such as constant darkness, but also give rise to more complex oscillatory phenomena including chaos and birhythmicity. By calculating bifurcations using Kawakami's method, we obtain detailed bifurcation diagrams related to stable and unstable invariant sets, and identify parameter regions in which the model generates complex oscillations as well as regular circadian oscillations. Moreover, we study bifurcations observed in the model incorporating the effect on a light-dark (LD) cycle and show that the waveform of the periodic variation in the light-induced parameter has a marked influence on the global bifurcation structure or the type of dynamic behavior resulting from the forcing term of the circadian oscillator by the LD cycles.     

The hepatic circadian clock is preserved in a lipid-induced mouse model of non-alcoholic steatohepatitis

Recent studies have correlated metabolic diseases, such as metabolic syndrome and non-alcoholic fatty liver disease, with the circadian clock. However, whether such metabolic changes per se affect the circadian clock remains controversial. To address this, we investigated the daily mRNA expression profiles of clock genes in the liver of a dietary mouse model of non-alcoholic steatohepatitis (NASH) using a custom-made, high-precision DNA chip. C57BL/6J mice fed an atherogenic diet for 5 weeks developed hypercholesterolemia, oxidative stress, and NASH. DNA chip analyses revealed that the atherogenic diet had a great influence on the mRNA expression of a wide range of genes linked to mitochondrial energy production, redox regulation, and carbohydrate and lipid metabolism. However, the rhythmic mRNA expression of the clock genes in the liver remained intact. Most of the circadianly expressed genes also showed 24-h rhythmicity. These findings suggest that the biological clock is protected against such a metabolic derangement as NASH.     

Clock polymorphisms and circadian rhythms phenotypes in a sample of the Brazilian population

A Clock polymorphism T to C situated in the 3' untranslated region (3'-UTR) has been associated with human diurnal preference. At first, Clock 3111C had been reported as a marker for evening preference. However these data are controversial, and data both corroborating and denying them have been reported. This study hypothesizes that differences in Clock genotypes could be observed if extreme morning-type subjects were compared with extreme evening-type subjects, and the T3111C and T257G polymorphisms were studied. The possible relationship between both polymorphisms and delayed sleep phase syndrome (DSPS) was also investigated. An interesting and almost complete linkage disequilibrium between the polymorphisms T257G in the 5' UTR region and the T3111C in the 3' UTR region of the Clock gene is described. Almost always, a G in position 257 corresponds to a C in position 3111, and a T in position 257 corresponds to a T in position 3111. The possibility of an interaction of these two regions in the Clock messenger RNA structure that could affect gene expression was analyzed using computer software. The analyses did not reveal an interaction between those two regions, and it is unlikely that this full allele correspondence affects Clock gene expression. These results show that there is no association between either polymorphism T3111C or T257G in the Clock gene with diurnal preference or delayed sleep phase syndrome (DSPS). These controversial data could result from the possible effects of latitude and clock genes interaction on circadian phenotypes.     

Synergy between the light-induced acute response and the circadian cycle: a new mechanism for the synchronization of the Phaseolus vulgaris clock to light

PvLHY and Lhcb expression has been studied in primary bean leaves after exposure of etiolated leaves to two or three white light-pulses and under different photoperiods. Under the tested photoperiods, the steady-state mRNA levels exhibit diurnal oscillations with zenith in the morning between ZT21 and 4 for PvLHY and between ZT4 and 6 for Lhcb. Nadir is in the evening between ZT12 and 18 for PvLHY and ZT18 and 24 for Lhcb. Light-pulses to etiolated seedlings induce a differentiated acute response that is reciprocally correlated with the amplitude of the following circadian cycle. In addition, the clock modulates the duration of the acute response (descending part of the curve included), which according to the phase of the rhythm at light application extends from 7 to 18 h. This constitutes the response dynamics of the Phaseolus clock to light. Similarly, the waveform of PvLHY and Lhcb expression during the day of different photoperiods resembles in induction capability (accomplishment of peak after lights-on) and duration (from lights-on phase to trough) the phase-dependent progression of acute response in etiolated seedlings. Consequently, the peak of Lhcb (all tested photoperiods) and PvLHY (in LD 18:6) attained in the photophase corresponds to the acute response peak, while the peak of PvLHY during the scotophase (in LD 12:12 and 6:18) corresponds to the circadian peak. Thus, the effect of the response dynamics in the photoperiod determines the coincidence of the peak with the photo- or scotophase, respectively. This represents a new model mechanism for the adaptation of the Phaseolus clock to light.     

Identification of a novel functional domain of ricin responsible for its potent toxicity

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved α-sarcin loop of large rRNAs. They have received attention in biological and biomedical research because of their unique biological activities toward animals and human cells as cell-killing agents. A better understanding of the depurination mechanism of RIPs could allow us to develop potent neutralizing antibodies and to design efficient immunotoxins for clinical use. Among these RIPs, ricin exhibited remarkable efficacy in depurination activity and highly conserved tertiary structure with other RIPs. It can be considered as a prototype to investigate the depurination mechanism of RIPs. In the present study, we successfully identified a novel functional domain responsible for controlling the depurination activity of ricin, which is located far from the enzymatic active site reported previously. Our study indicated that ricin A-chain mAbs binding to this domain (an α-helix comprising the residues 99-106) exhibited an unusual potent neutralizing ability against ricin in vivo. To further investigate the potential role of the α-helix in regulating the catalytic activity of ricin, ricin A-chain variants with different flexibility of the α-helix were rationally designed. Our data clearly demonstrated that the flexibility of the α-helix is responsible for controlling the depurination activity of ricin and determining the extent of protein synthesis inhibition, suggesting that the conserved α-helix might be considered as a potential target for the prevention and treatment of RIP poisoning.     

Identification of a non-basic domain in the histone H4 N-terminus required for repression of the yeast silent mating loci.

We have shown previously that a stretch of four charged residues (16-19) at the histone H4 N-terminus is involved in repression of the yeast silent mating loci. One of these residues, Lys16, is a site for acetylation, which may prevent repression of the silent mating loci. In this paper we ask whether other sequences in histone H4, possibly in conjunction with H3 residues, are required for repression. We find that even in combination, the other seven acetylatable lysines in H3 and H4 do not function in repression. In contrast, we have found that an adjacent relatively uncharged domain (residues 21-29) is required for repression and that single amino acid insertions and deletions in this region are extremely detrimental. We propose that the basic and non-basic domains together form a DNA (or protein) induced amphipathic alpha-helix required in the formation of a repressive chromatin structure.     

Identification of a novel protein for memory regulation in the hippocampus

Memory formation, maintenance, and retrieval are a dynamic process, reflecting a combined outcome of new memory formation on one hand, and older memory suppression/clearance on the other. Although much knowledge has been gained regarding new memory formation, less is known about the molecular components and processes that serve the function of memory suppression/clearance. Here, we report the identification of a novel protein, termed hippyragranin (HGN), that is expressed in the rat hippocampus and its expression is reduced by hippocampal denervation. Inhibition of HGN by antisense oligonucleotide in area CA1 results in enhanced performance in Morris water maze, as well as elevated long-term potentiation. These results suggest that HGN is involved in negative memory regulation.     

Identification of a novel dynein binding domain in nudel essential for spindle pole organization in Xenopus egg extract

The nuclear distribution protein E (NudE) and nuclear distribution protein E-like (Nudel or Ndel1) interact with both lissencephaly 1 (Lis1) and dynein. These interactions are thought to be essential for dynein function. Previous studies have shown that the highly conserved N terminus of NudE/Nudel directly binds to Lis1, and such binding is critical for dynein activity. By contrast, although the C terminus of NudE/Nudel was reported to bind to dynein, the functional significance of this binding has remained unclear. Using the sperm-mediated spindle assembly assay in Xenopus egg extracts and extensive mutagenesis studies, we have identified a highly conserved dynein binding domain within the first 80 amino acids of Nudel. We further demonstrate that the dynein intermediate chain in the dynein complex is directly involved in this interaction. Importantly, we show that both the dynein and Lis1 binding domains of Nudel are required for spindle pole organization. Finally, we report that spindle defects caused by immuno-depletion of Nudel could be rescued by a 1-fold increase of Lis1 concentration in Xenopus egg extracts. This suggests that an important function of the N terminus of Nudel is to facilitate the interaction between Lis1 and dynein during spindle assembly. Together, our findings open up new avenues to further decipher the mechanism of dynein regulation by Nudel and Lis1.     

Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.

A human orthologue of the Saccharomyces cerevisiae YVH1 protein-tyrosine phosphatase is able to rescue the slow growth defect caused by the disruption of the S. cerevisiae YVH1 gene. The human YVH1 gene is located on chromosome 1q21-q22, which falls in a region amplified in human liposarcomas. The evolutionary conserved COOH-terminal noncatalytic domain of human YVH1 is essential for in vivo function. The cysteine-rich COOH-terminal domain is capable of coordinating 2 mol of zinc/mol of protein, defining it as a novel zinc finger domain. Human YVH1 is the first protein-tyrosine phosphatase that contains and is regulated by a zinc finger domain.