CLOCK、BMAL1半胱氨酸残基氧化对生物节律的影响及机制研究

Unravelling the mechanism of circadian rhythm disturbances induced by space environmental factors is significant and beneficial to the .health maintenance of astronauts. The transcriptional regulation mechanism and the non-transcriptional regulation mechanism are two molecular mechanisms of circadian rhythms, However, the intrinsic link between the two mechanisms is unclear. The oxidative modification of cysteine residue is a vital modification of post-translational modification of proteins. In recent years, studies found that a series of important proteins are subject to cysteine oxidative modification in vivo, and this modification has important role in biological process. Our preliminary research found that biorhythms associated proteinCLOCK and BMAL1 cysteine residues oxidation was present and showed rhythmicity in vivo. CLOCK and BMAL1 cysteine oxidation may mediate the interaction of transcriptional mechanisms and non-transcriptional mechanisms.This project aims to use site-directed mutagenesis method to determine the site of cysteine which was oxidative modified, use luciferase reporter gene experiments to test the effects of cysteine oxidative modification on cell circadian rhythm, decipher the mechanisms of the effects of cysteine oxidative modification on cell circadian rhythm from subcellular localization of CLOCK and BMAL1, synthesis and degradation, transcriptional activity aspect, Finally, we will explore the specific cysteine modification of CLOCK and BMAL1 under the stimulation of H2O2 clinorotation simulated microgravity and their influence on cell circadian rhythm and the protective effects of NAC and GSH.

对航天环境因素引起的生物节律紊乱发生机制的深入研究对于保障航天员的健康具有重要意义。转录机制和非转录机制是生物体昼夜节律产生的两种分子机制，然而，这两种机制之间的内在联系还不清楚。蛋白质Cys残基的氧化修饰是一种重要的蛋白质翻译后修饰过程，体内一系列重要蛋白质的Cys均受到氧化修饰调节，且这种调控方式影响了体内重要的生物过程。前期研究发现生物节律相关蛋白CLOCK、BMAL1的Cys残基均可发生氧化并具有节律性。CLOCK、BMAL1 Cys氧化调节可能介导了转录机制和非转录机制之间的相互作用。本研究拟利用定点突变的方法验证CLOCK、BMAL1 Cys发生氧化修饰的特定位点，运用荧光素酶报告基因实验验证特定位点Cys氧化修饰对细胞自身节律的影响，并研究在H2O2、回转模拟微重力刺激下CLOCK、BMAL1 Cys氧化修饰的变化及对细胞节律的影响，及NAC、GSH等抗氧化物质的保护作用。

转录机制和非转录机制是生物节律产生的两种分子机制，然而，这两种机制之间的内在联系还不清楚。有报道表明，氧化应激是影响生物节律的重要因素，然而，到目前 为止ROS对节律基因表达的调控作用还不明确。本课题研究了节律周期内CLOCK、BMAL1 Cys的氧化，过氧化氢对CLOCK、BMAL1，PER1、PER2，CRY1、CRY2等节律基因相关蛋白表达的影响及其机制；过氧化氢对CLOCK、BMAL1出入核的影响及抗氧化物质NAC对过氧化氢引起的CLOCK、BMAL1表达上调的改善作用；此外，我们还研究了回转模拟微重力效应对CLOCK、BMAL1表达的作用。实验结果表明，CLOCK、BMAL1 Cys的氧化均表现为24小时节律性，Cys位点突变可引起CLOCK、BMAL1转录活性的下降，过氧化氢可导致CLOCK 、BMAL1蛋白质表达节律的紊乱并阻碍CLOCK 、BMAL1的细胞核转位；过氧化氢上调节律基因CLOCK、BMAL1、PER1、PER2、CRY1、CRY2的表达，核受体NR1D1、NR1D2 、RORa参与了过氧化氢对CLOCK、BMAL1表达的调控作用，NAC可改善过氧化氢引起的CLOCK、BMAL1表达上调；回转模拟微重力效应对CLOCK、BMAL1表达调控具有与过氧化氢相似的作用。本研究为揭示氧化应激及航天微重力对生物节律的影响及相关机制提供了实验基础