蓝藻生物节律性分子调控机制的研究进展

生物钟现象是一种普遍存在于生物界细胞的内源节律性保持机制。生物钟机制的存在可以使生物体的代谢行为产生并维持以24 h为周期的昼夜节律,从而更好地适应于地球自转所产生的环境条件昼夜间节律性变化。蓝藻是目前生物钟分子机制研究中的模式生物,其依赖于k ai基因家族成员的核心生物钟调控模式已经被众多研究者详细阐明。蓝藻生物钟的核心振荡器是由蓝藻k aiA/B/C的编码产物来调控的,Kai蛋白的表达模式具有节律性。KaiC蛋白磷酸化状态的节律性循环及输入、输出途径相关组成蛋白的翻译后修饰状态节律性循环共同组成其反馈回路,负责维持生物钟节律性振荡的持续进行并与环境周期保持同步。传统的蓝藻生物钟分子机制模型认为,节律性表达基因翻译产物的转录/翻译负反馈抑制环是生物节律性维持和输出的关键。遗憾的是,在其它物种生物钟分子机制研究中未发现由kai基因家族成员同源基因组成的节律性标签,这表明以k aiA/B/C为核心振荡器的生物钟系统并不是一种跨物种保守的生物钟系统。近期,人们发现非转录/翻译依赖的振荡器(NTO)也具有成为生物节律性产生和维持的“源动力”的可能。过氧化物氧化还原酶(PRX)氧化还原状态节律性是第一种被报道的跨物种保守的NTO节律性标签,这也日渐成为蓝藻生物钟分子机制研究新的热点。     

铜绿微囊藻生物钟蛋白KaiA的自身相互作用研究

正蓝藻虽为原核生物,但它也和真核生物一样具有生物钟,它的固氮作用、光合作用、氨基酸吸收、细胞分裂以及基因表达等生理代谢过程都受到生物钟的调控,具有昼夜节律性。虽然蓝藻生物钟和真核生物钟一样,都以近24h的周期运行,都具有温度补偿效应,光、温等环境因素都能重置生物钟的时相,但组成蓝藻生物钟的钟蛋白与真核生物钟蛋白间不具有任何同源性,蓝藻生物钟的计时机制也与真核生物钟存在差异1-2。蓝藻钟基因为一个基因簇kai,由三个基因kaiA、kaiB、kaiC以单一拷贝成簇排列,Kai蛋白组成蓝藻生物钟的核心即中央振荡器,其中kaiC蛋白的磷酸化状态是中央振荡器产生周期性震荡的关键,它决定中央振荡器的时相,而kaiC的磷酸化状态则受到kaiA和kaiB的调节。kaiA是接受和整合环境信息的钟蛋白,具有N-端和C-端两个结构域,N-端缺乏保守天冬氨酰残基的伪接受域能通过与输入途径的某种蛋白(目前未知)发生相互作用而感受环境信号       

KaiA调控蓝藻生物钟的分子机制研究进展

蓝藻生物钟系统主要包括输入途径、核心振荡器和输出途径3部分,核心振荡器主要由时钟蛋白KaiA、KaiB、KaiC构成。3种蛋白之间的相互作用产生节律信号及调控输入、输出信号进而维持生物振荡的精确与稳定。文中围绕蓝藻生物钟核心振荡器及核心振荡器组成蛋白的结构、功能与相互作用特点,结合本实验室近期取得的研究成果,针对时钟蛋白KaiA调节KaiC的酶活性、介导核心振荡器的时相重置、与CikA竞争KaiB的结合位点等方面近年来的研究进展进行了综述。     

生物节律基因period3的研究进展

昼夜节律是所有真核生物和部分原核生物的基本特征,一组节律表达的生物钟基因形成24 h周期振荡的自主调节转录-翻译反馈回路。period（per）基因家族是生物钟反馈回路中重要组成成分,per3基因是period基因家族成员之一。人类的per3基因定位于染色体1p36,其编码区第18外显子中含有一个灵长类特有的串联重复序列（variable number tandem repeat,VNTR）。该VNTR包含一簇理论上的磷酸化位点,能影响PER3蛋白的磷酸化降解,影响PER3蛋白的功能。近年研究发现,per3基因多态性与睡眠结构、睡眠紊乱发病年龄、睡眠剥夺后次日清晨执行能力等密切相关。     

O-GlcNAc修饰调节生物节律研究进展

生物体的睡眠/觉醒、进食等行为以及各种生理、生化、代谢过程都遵循着大约24 h的周期性变化,称为昼夜节律(circadian rhythms)。昼夜节律与能量代谢之间存在着紧密的联系。位于下丘脑视交叉上核(suprachiasmatic nuclei,SCN)的中枢生物钟与外周组织细胞中的生物钟共同组成了哺乳动物的昼夜节律系统。以CLOCK/BMAL1异二聚体为核心的转录/翻译负反馈环保障了节律系统的正常运行。各种蛋白质翻译后修饰参与了昼夜节律的调控。综述了氧连β-N-乙酰葡糖胺修饰(O-Glc NAcylation)在调节昼夜节律中发挥的重要作用。O-Glc NAc修饰可以增强一些生物钟蛋白的稳定性及转录活性,也可以影响其他一些生物钟蛋白的磷酸化及细胞定位。抑制生物钟蛋白的O-Glc NAc修饰导致细胞节律衰弱和多种节律基因表达下调。研究表明,O-Glc NAc作为机体能量代谢的感受器参与了多条细胞代谢相关信号转导通路的调节,O-Glc NAc修饰为能量代谢影响昼夜节律提供了一条新的途径。     

RNA甲基化依赖的RNA加工调控昼夜节律生物钟的速度

<正>哺乳动物的昼夜节律生物钟主要通过调控代谢开关或限速酶的表达来调节新陈代谢。真核生物的生物钟包括一个转录-翻译负反馈调节通路,通过这个通路,生物钟基因调节它们自身以及重要代谢基因的表达。多年前人们就已知道肝脏中大约10%的基因具有昼夜节律性,而近些年的RNA测序研究表明,在这些节律性的基因中,仅有大约五分之一是由从头转录所驱使的。这一发现提示对RNA剪接和加工的调控具有非常重要的生物学意义。     

昆虫生物钟分子调控研究进展

昆虫生物钟节律的研究是人类了解生物节律的重要途径。昆虫在生理和行为上具有广泛的节律活动,如运动、睡眠、学习记忆、交配、嗅觉等节律活动,其中昼夜活动行为节律的研究广泛而深入。昆虫乃至高等动物普遍具有保守的昼夜节律系统,昼夜生物钟节律主要包括输入系统:用于接受外界光和温度等环境信号并传入核心振荡器,使得生物时钟与环境同步;核心时钟系统:自我维持的昼夜振荡器;输出系统:将生物钟产生的信号传递出去而控制生物行为和生理的节律变化。早期分子和遗传学研究主要关注昼夜节律振荡器的分子机制及神经生物学,阐明了昼夜生物钟节律的主要分子机制及相关神经网络。最近更多的研究关注生物钟信号是如何输入和输出。本文以果蝇运动节律的相关研究为主要内容,围绕生物钟输入系统、振荡器、输出系统这3个组成部分对昆虫生物钟研究进展进行总结。     

生物钟基因系统功能异常与心血管疾病和消化疾病的关联性机制研究

生物体内源性生物钟产生的昼夜节律是以近24 h的节律性振荡对外界环境变化进行的综合性调节反应,其产生的分子基础是生物钟基因及其编码的蛋白质组成的转录-翻译反馈环路,其中生物钟基因可作用于下游钟控基因而调节机体各项生理功能。昼夜节律紊乱、生物钟基因表达改变,与许多疾病包括心血管疾病和消化疾病的发生发展相关,甚至是癌症发生的重要促进因素。对昼夜节律的研究为疾病的预防和治疗提供了新思路。     

拟南芥生物钟分子机制研究进展

本文主要概述了目前拟南芥生物钟分子机制的研究进展.生物钟通过调控导引节律的相位来调节植物的生理活动.拟南芥生物钟由CCAJ、LHy和TOCJ 3个主要基因构成了一个稳定的负反馈环,来调节昼夜节律中各个基因如APRR/TOC15重奏的作用,从而调控昼夜节律的相位.在开花的光周期调控中,提出了外协和模型,其中的关键基因是CO,它与拟南芥的开花时间直接相关.     

血压昼夜节律特征及其分子调控机制

正常血压具有典型的昼夜节律特征。血压昼夜节律异常与高血压靶器官损害和心血管事件发生呈明显相关关系,是独立于血压水平的重要致病因素。血压昼夜节律的产生和维持与时钟基因的周期性表达有关。时钟基因bmal1、per2是体内生物钟系统运行的关键基因,其表达水平和节律变化直接调节血压的昼夜节律。     

In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB

Biochemical circadian oscillation of KaiC phosphorylation, by mixing three Kai proteins and ATP, has been proven to be the central oscillator of the cyanobacterial circadian clock. In vivo, the intracellular levels of KaiB and KaiC oscillate in a circadian fashion. By scrutinizing KaiC phosphorylation rhythm in a wide range of Kai protein concentrations, KaiA and KaiB were found to be “parameter-tuning” and “state-switching” regulators of KaiC phosphorylation rhythm, respectively. Our results also suggest a possible entrainment mechanism of the cellular circadian clock with the circadian variation of intracellular levels of Kai proteins.     

Quantifying the Rhythm of KaiB-C Interaction for In Vitro Cyanobacterial Circadian Clock

An oscillator consisting of KaiA, KaiB, and KaiC proteins comprises the core of cyanobacterial circadian clock. While one key reaction in this process-KaiC phosphorylation-has been extensively investigated and modeled, other key processes, such as the interactions among Kai proteins, are not understood well. Specifically, different experimental techniques have yielded inconsistent views about Kai A, B, and C interactions. Here, we first propose a mathematical model of cyanobacterial circadian clock that explains the recently observed dynamics of the four phospho-states of KaiC as well as the interactions among the three Kai proteins. Simulations of the model show that the interaction between KaiB and KaiC oscillates with the same period as the phosphorylation of KaiC, but displays a phase delay of ～8 hr relative to the total phosphorylated KaiC. Secondly, this prediction on KaiB-C interaction are evaluated using a novel FRET (Fluorescence Resonance Energy Transfer)-based assay by tagging fluorescent proteins Cerulean and Venus to KaiC and KaiB, respectively, and reconstituting fluorescent protein-labeled in vitro clock. The data show that the KaiB∶KaiC interaction indeed oscillates with ～24 hr periodicity and ～8 hr phase delay relative to KaiC phosphorylation, consistent with model prediction. Moreover, it is noteworthy that our model indicates that the interlinked positive and negative feedback loops are the underlying mechanism for oscillation, with the serine phosphorylated-state (the "S-state") of KaiC being a hub for the feedback loops. Because the kinetics of the KaiB-C interaction faithfully follows that of the S-state, the FRET measurement may provide an important real-time probe in quantitative study of the cyanobacterial circadian clock.     

A kaiC-interacting sensory histidine kinase, SasA, necessary to sustain robust circadian oscillation in cyanobacteria

Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms.     

KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB and KaiC proteins are essential for generation of circadian rhythms. We quantitatively analyzed the intracellular dynamics of these proteins and found a circadian rhythm in the membrane/cytosolic localization of KaiB, such that KaiB interacts with a KaiA-KaiC complex during the late subjective night. KaiB-KaiC binding is accompanied by a dramatic reduction in KaiC phosphorylation and followed by dissociation of the clock protein complex(es). KaiB attenuated KaiA-enhanced phosphorylation both in vitro and in vivo. Based on these results, we propose a novel role for KaiB in a regulatory link among subcellular localization, protein-protein interactions and post-translational modification of Kai proteins in the cyanobacterial clock system.     

Cyanobacterial circadian clockwork: roles of KaiA,KaiB and the kaiBC promoter in regulating KaiC

Using model strains in which we ectopically express the cyanobacterial clock protein KaiC in cells from which the clock genes kaiA, kaiB and/or kaiC are deleted, we found that some features of circadian clocks in eukaryotic organisms are conserved in the clocks of prokaryotic cyanobacteria, but others are not. One unexpected difference is that the circadian autoregulatory feedback loop in cyanobacteria does not require specific clock gene promoters as it does in eukaryotes, because a heterologous promoter can functionally replace the kaiBC promoter. On the other hand, a similarity between eukaryotic clock proteins and the cyanobacterial KaiC protein is that KaiC is phosphorylated in vivo. The other essential clock proteins KaiA and KaiB modulate the status of KaiC phosphorylation; KaiA inhibits KaiC dephosphorylation and KaiB antagonizes this action of KaiA. Based upon an analysis of clock mutants, we conclude that the circadian period in cyanobacteria is determined by the phosphorylation status of KaiC and also by the degradation rate of KaiC. These observations are integrated into a model proposing rhythmic changes in chromosomal status.     

Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro

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.     

Synchronization of Circadian Oscillation of Phosphorylation Level of KaiC In Vitro

In recent experimental reports, robust circadian oscillation of the phosphorylation level of KaiC has been reconstituted by incubating three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro. This reconstitution indicates that protein-protein interactions and the associated ATP hydrolysis suffice to generate the oscillation, and suggests that the rhythm arising from this protein-based system is the circadian clock pacemaker in cyanobacteria. The mechanism of this reconstituted oscillation, however, remains elusive. In this study, we extend our previous model of oscillation by explicitly taking two phosphorylation sites of KaiC into account and we apply the extended model to the problem of synchrony of two oscillatory samples mixed at different phases. The agreement between the simulated and observed data suggests that the combined mechanism of the allosteric transition of KaiC hexamers and the monomer shuffling between them plays a key role in synchronization among KaiC hexamers and hence underlies the population-level oscillation of the ensemble of Kai proteins. The predicted synchronization patterns in mixtures of unequal amounts of two samples provide further opportunities to experimentally check the validity of the proposed mechanism. This mechanism of synchronization should be important in vivo for the persistent oscillation when Kai proteins are synthesized at random timing in cyanobacterial cells.