The daily rhythm of mice

The house mouse Mus musculus represents a valuable tool for the analysis and the understanding of the mammalian circadian oscillator. Forward and reverse genetics allowed the identification of clock components and the verification of their function within the circadian clockwork. In many cases unforeseen links were discovered between a particular circadian regulatory protein and various diseases or syndromes. Thus, this model system is not only perfectly suited to pinpoint the components of the mammalian circadian clock, but also to unravel metabolic, physiological, and pathological processes linked to the circadian timing system.     

Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.     

Circadian proteomics of the mouse retina

The circadian clock in the retina regulates a variety of physiological phenomena such as disc shedding and melatonin release. Although these events are critical for retinal functions, it is almost unknown how the circadian clock controls the physiological rhythmicity. To gain insight into the processes, we performed a proteomic analysis using 2-DE to find proteins whose levels show circadian changes. Among 415 retinal protein spots, 11 protein spots showed circadian rhythmicity in their intensities. We performed MALDI-TOF MS and NanoLC-MS/MS analyses and identified proteins contained in the 11 spots. The proteins were related to vesicular transport, calcium-binding, protein degradation, metabolism, RNA-binding, and protein foldings, suggesting the clock-regulation of neurotransmitter release, transportation of the membrane proteins, calcium-binding capability, and so on. We also found a rhythmic phosphorylation of N-ethylmaleimide-sensitive fusion protein and identified one of the amino acid residues modified by phosphorylation. These findings provide a new perspective on the relationship between the physiological functions of the retina and the circadian clock system.     

Shutoff of histone synthesis by Mengovirus infection of Ehrlich ascites tumor cells.

The histone synthesizing capacity of mengovirus-infected Ehrlich ascites tumor cells and of their corresponding postnuclear supernatants was investigated as a funcion of time post-infection. In addition, histone synthesis was compared with the synthesis of other basic host proteins under identical conditions. In the scope of mengovirus infection of Ehrlich ascites tumor cells the less complex fraction comprising basic protein, separated from the acidic proteins by carboxymethyl cellulose chromatography, can be regarded as a representative of total host protein. Histones and the remaining basic host proteins therefore are well suited as easily identifiable indicators of the host protein synthesizing potential of mengovirus-infected Ehrlich ascites tumor cells. The cessation of histone synthesis proceeds faster than the arrest of the synthesis of other basic host protein.     

Protein biomarker enrichment by biomarker antibody complex elution for immunoassay biosensing

It is very challenging to perform sample enrichment for protein biomarkers because proteins can easily change conformation and denature. In this paper we demonstrate protein enrichment suited for high-sensitivity integrated immuno-biosensing. The method enhances the concentration of the biomarkers and simultaneously removes matrix components that could interfere with the immunoassay. Biomarkers are captured using antibody coated magnetic particles and the biomarker antibody complexes are released by enzymatic elution. The eluted complexes are subsequently detected in a sandwich immunoassay biosensor. A scaling study of the enrichment process demonstrates an enrichment factor of 15 in buffer and plasma. We analyze the enrichment factor in terms of the three basic steps of the assay (capture, concentration, elution) and we quantify their respective efficiencies. The process is suited for integration into bio-analytical tools.     

Dynamic proteomic analysis reveals diurnal homeostasis of key pathways in rice leaves

Diurnal physiological acclimation regulated by a circadian system is an advantage for plant fitness. The circadian system is composed of a signal input, the clock and output pathways. Understanding the regulation mechanism of the output pathways remains a major challenge. Diurnal proteomic change reflects the state of circadian organization. We found the content of glucose, fructose, sucrose and starch diurnally changed in leaves of rice seedlings grown under a 12-h light/12-h dark condition with constant temperature. Dynamic proteomics analysis revealed 140 protein spots with diurnally changed levels at six times of the light/dark cycle; 132 spots were identified by MS, and 119 spots were of a single protein each with functional annotation. These proteins are involved in regulation of carbohydrate flow, redox, protein folding, nitrogen and protein metabolism, energy conversion, photorespiration and photosynthesis. Of these proteins, 81.5% were upregulated during the light phase, overlappingly, 41.2% showed behavior of circadian anticipation to dawn. Pattern analysis showed that the diurnal regulation involved pathways of allocation of carbohydrates between temporary reserves and consumption, maintenance of redox homeostasis, diurnal protein reassembly and nitrogen assimilation. These pathways reflect biochemical phenotypes of the circadian change linking the oscillator and circadian outputs.     

Circadian formation of clock protein complexes by KaiA,KaiB, KaiC,and SasA in cyanobacteria

Physical interactions among clock-related proteins KaiA, KaiB, KaiC, and SasA are proposed to be important for circadian function in the cyanobacterium Synechococcus elongatus PCC 7942. Here we show that the Kai proteins and SasA form heteromultimeric protein complexes dynamically in a circadian fashion. KaiC forms protein complexes of approximately 350 and 400-600 kDa during the subjective day and night, respectively, and serves as a core of the circadian protein complexes. This change in the size of the KaiC-containing complex is accompanied by nighttime-specific interaction of KaiA and KaiB with KaiC. In various arrhythmic mutants that lack each functional Kai protein or SasA, circadian rhythms in formation of the clock protein complex are abolished, and the size of the protein complexes is dramatically affected. Thus, circadian-regulated formation of the clock protein complexes is probably a critical process in the generation of circadian rhythm in cyanobacteria.     

Two circadian timing circuits in Neurospora crassa cells share components and regulate distinct rhythmic processes

In Neurospora crassa, FRQ, WC-1, and WC-2 proteins comprise the core circadian FRQ-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. However, physiological and biochemical studies have demonstrated the existence of additional oscillators in the cell that function in the absence of FRQ (collectively termed FRQ-less oscillators [FLOs]). Whether or not these represent temperature-compensated, entrainable circadian oscillators is not known. The authors previously identified an evening-peaking gene, W06H2 (now called clock-controlled gene 16 [ccg-16]), which is expressed with a robust daily rhythm in cells that lack FRQ protein, suggesting that ccg-16 is regulated by a FLO. In this study, the authors provide evidence that the FLO driving ccg-16 rhythmicity is a circadian oscillator. They find that ccg-16 rhythms are generated by a temperature-responsive, temperature-compensated circadian FLO that, similar to the FRQ-based oscillator, requires functional WC-1 and WC-2 proteins for activity. They also find that FRQ is not essential for rhythmic WC-1 protein levels, raising the possibility that this WCFLO is involved in the generation of WC-1 rhythms. The results are consistent with the presence of 2 circadian oscillators within Neurospora cells, which the authors speculate may interact with each other through the shared WC proteins.     

Functional proteomics: a promising approach to find novel components of the circadian system

In the postgenome era, the analysis of entire subproteomes in correlation with their function has emerged due to high throughput technologies. Early approaches have been initiated to identify novel components of the circadian system. For example, in the marine dinoflagellate Lingulodinium polyedra, a chronobiological proteome assay was performed, which resulted in the identification of already known circadian expressed proteins as well as novel temporal controlled proteins involved in metabolic pathways. In the green alga Chlamydomonas reinhardtii, two circadian expressed proteins (a protein disulfide isomerase and a tetratricopeptide repeat protein) were identified by functional proteomics. Also, the first hints of temporal control within chloroplast proteins of Arabidopsis thaliana were identified by proteome analysis.     

Antibodies for assessing circadian clock proteins in the rodent suprachiasmatic nucleus

Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry.     

Coupling of human circadian and cell cycles by the timeless protein

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. We have found that the human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein.     

Functional Proteomics: A Promising Approach to Find Novel Components of the Circadian System

In the postgenome era, the analysis of entire subproteomes in correlation with their function has emerged due to high throughput technologies. Early approaches have been initiated to identify novel components of the circadian system. For example, in the marine dinoflagellate Lingulodinium polyedra, a chronobiological proteome assay was performed, which resulted in the identification of already known circadian expressed proteins as well as novel temporal controlled proteins involved in metabolic pathways. In the green alga Chlamydomonas reinhardtii, two circadian expressed proteins (a protein disulfide isomerase and a tetratricopeptide repeat protein) were identified by functional proteomics. Also, the first hints of temporal control within chloroplast proteins of Arabidopsis thaliana were identified by proteome analysis.     

Proteins found in a CikA interaction assay link the circadian clock, metabolism, and cell division in Synechococcus elongatus

Diverse organisms time their cellular activities to occur at distinct phases of Earth's solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short circadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions--circadian period regulation, phase resetting, and cell division--attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.