KaiA, KaiB, and KaiC are essential proteins of the circadian clock in the cyanobacterium Synechococcus elongatus PCC 7942. The phosphorylation cycle of KaiC that occurs in vitro after mixing the three proteins and ATP is thought to be the master oscillation governing the circadian system. We analyzed the temporal profile of complexes formed between the three Kai proteins. In the phosphorylation phase, KaiA actively and repeatedly associated with KaiC to promote KaiC phosphorylation. High levels of phosphorylation of KaiC induced the association of the KaiC hexamer with KaiB and inactivate KaiA to begin the dephosphorylation phase, which is closely linked to shuffling of the monomeric KaiC subunits among the hexamer. By reducing KaiC phosphorylation, KaiB dissociated from KaiC, reactivating KaiA. We also confirmed that a similar model can be applied in cyanobacterial cells. The molecular model proposed here provides mechanisms for circadian timing systems.
In the cyanobacterium Synechococcus elongatus PCC 7942, KaiA, KaiB, and KaiC are essential proteins for the generation of a circadian rhythm. KaiC is proposed as a negative regulator of the circadian expression of all genes in the genome, and its phosphorylation is regulated positively by KaiA and negatively by KaiB and shows a circadian rhythm in vivo. To study the functions of KaiC phosphorylation in the circadian clock system, we identified two autophosphorylation sites, Ser-431 and Thr-432, by using mass spectrometry (MS). We generated Synechococcus mutants in which these residues were substituted for alanine by using site-directed mutagenesis. Phosphorylation of KaiC was reduced in the single mutants and was completely abolished in the double mutant, indicating that KaiC is also phosphorylated at these sites in vivo. These mutants lost circadian rhythm, indicating that phosphorylation at each of the two sites is essential for the control of the circadian oscillation. Although the nonphosphorylatable mutant KaiC was able to form a hexamer in vitro, it failed to form a clock protein complex with KaiA, KaiB, and SasA in the Synechococcus cells. When nonphosphorylatable KaiC was overexpressed, the kaiBC promoter activity was only transiently repressed. These results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins.
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 ϳ350 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.
The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three purified clock proteins, KaiA, KaiB and KaiC, with ATP. The KaiC phosphorylation rhythm persists for at least 10 days without damping. By mixing oscillatory samples that have different phases and analyzing the dynamics of their phase relationships, we found that the robustness of the KaiC phosphorylation rhythm arises from the rapid synchronization of the phosphorylation state and reaction direction (phosphorylation or dephosphorylation) of KaiC proteins. We further demonstrate that synchronization is tightly linked with KaiC dephosphorylation and is mediated by monomer exchange between KaiC hexamers during the early dephosphorylation phase. This autonomous synchronization mechanism is probably the basis for the resilience of the cyanobacterial circadian system against quantitative fluctuations in clock components during cellular events such as cell growth and division.
Prolonged exposure to ultraviolet (UV) radiation causes photoaging of the skin and induces a number of disorders, including sunburn, fine and coarse wrinkles, and skin cancer risk. Therefore, the application of sunscreen has gained much attention to reduce the harmful effects of UV irradiation on our skin. Recently, there has been a growing demand for the replacement of chemical sunscreens with natural UV-absorbing compounds. Mycosporine-like amino acids (MAAs), promising alternative natural UV-absorbing compounds, are a group of widely distributed, low molecular-weight, water-soluble molecules that can absorb UV radiation and disperse the absorbed energy as heat, without generating reactive oxygen species (ROS). More than 30 MAAs have been characterized, from a variety of organisms. In addition to their UV-absorbing properties, there is substantial evidence that MAAs have the potential to protect against skin aging, including antioxidative activity, anti-inflammatory activity, inhibition of protein-glycation, and inhibition of collagenase activity. This review will provide an overview of MAAs, as potential anti-aging ingredients, beginning with their structure, before moving on to discuss the most recent experimental observations, including the molecular and cellular mechanisms through which MAAs might protect the skin. In particular, we focus on the potential anti-aging activity of mycosporine-2-glycine (M2G).
Effects of natural catalysts, isothiocyanates and polysulfides, on Z-isomerization and decomposition of (all-E)carotenoids (lycopene, β-carotene, and astaxanthin) after heat treatment were investigated. When isothiocyanates were added to (all-E)-carotenoid solutions and heated, Z-isomerization and decomposition of carotenoids were enhanced and the degree differed depending on the isothiocyanate type. Interestingly, when polysulfides were applied in the same manner, in addition to promoting the Z-isomerization reaction, they markedly improved the thermal stability of carotenoids. Successively, we investigated the reaction characteristics of allyl isothiocyanate (AITC) and diallyl disulfide (DADS) using (all-E)-lycopene; that is, effects of the amount added, solvent used, and reaction temperature and time, as well as the combination use on Z-isomerization and decomposition of lycopene, were investigated. With increases in the amount added and reaction temperature and time, Z-isomerization of lycopene was promoted for both catalysts. The high-temperature treatment tests clearly showed that AITC induced thermal decomposition of lycopene, whereas DADS improved the lycopene stability. Moreover, the simultaneous use of AITC and DADS resulted in a synergetic effect on the Z-isomerization efficiency.
The three cyanobacterial Kai proteins and ATP are capable of generating an autonomous rhythm of KaiC phosphorylation in a test tube. As the period is Ϸ24 hours and is stable in a wide temperature range, this rhythm is thought to function as the basic oscillator of the cyanobacterial circadian system. We have examined the rhythm under various temperature cycles and found that it was stably entrained by a temperature cycle of 20 -28 hours. As the period length was not altered by temperature, entrainment by period change could be excluded from possible mechanisms. Instead, temperature steps between 30°and 45°C and vice versa shifted the phase of the rhythm in a phase-dependent manner. Based on the phase response curves of the step-up and step-down in temperature, phase shift by single temperature pulse was estimated using a nonparametric entrainment model (discontinuous phase jump by external stimuli). The predicted phase shift was consistent with the experimentally measured phase shift. Next, successive phase shifts caused by repeated temperature cycles were computed by two phase response curves and compared with actual entrainment of the rhythm. As the entrainment pattern observed after various combinations of temperature cycles matched the prediction, it is likely that nonparametric entrainment functions even in the simple three-protein system. We also analyzed entrainment of KaiC phosphorylation by temperature cycle in cyanobacterial cells and found both the parametric and the nonparametric models function in vivo.circadian clock ͉ KaiC phosphorylation rhythm ͉ Phase response ͉ temperature entrainment
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