Degradation Mechanisms of Mammalian Circadian Clock Proteins

描述（由申请人提供）：生物钟几乎影响哺乳动物生命的所有方面，使我们的内部生理过程与一天中的最佳时间保持一致。了解保持昼夜节律的分子电路可以让我们深入了解生物钟如何驱动明显的节奏，以及当昼夜节律系统被打乱时（即在轮班工作期间）该如何修复。在负反馈循环系统中，昼夜节律时间编码于“时钟基因”表达的节律调节中。这个定时系统的关键是丰富的生物钟蛋白的昼夜降解；然而，这些机制仍然难以捉摸。为了开始阐明这些机制，我们开发了一种新的功能筛选方法来确定哪些E3泛素连接酶会降解哪些时钟蛋白，并筛选了降解RevErb / o蛋白的E3连接酶。这两种RevErb蛋白都是正常生物钟功能所必需的，并表现出高振幅的丰度节律，但驱动其昼夜节律清除的机制尚不清楚。我们的筛选发现了两个新的候选RevErb E3连接酶Spsb4和Siah2，初步数据表明这些E3s直接参与了RevErb蛋白的节律性降解。此外，我们的数据首次表明，昼夜混响退化的速度是昼夜节律周期性的决定因素。确定Spsb4和Siah2在整体时钟功能中的作用、机制和贡献是我们当前研究计划的主要重点。由于这个屏幕上的两个命中似乎都是混响稳定性的真正监管机构，我们提议的另一个重点是扩大我们的筛选工作。在Specific Aim 1中提出的实验重点是阐明振荡细胞系统中Siah2/Spsb4降解RevErb蛋白的机制。在Specific Aim 2中提出的实验更深入地探讨了siah2介导的RevErb蛋白降解在体内整体时钟功能中的作用。在Specific Aim 3中的实验将使用扩展的E3连接酶cDNA筛选文库来鉴定所有剩余必需核心时钟蛋白的候选E3。这些目标的成功将为我们未来确定蛋白质降解机制的努力提供必要的背景和验证。总之，揭示介导时钟蛋白节律性降解的新机制将为治疗昼夜节律相关疾病开辟许多新途径。

DESCRIPTION (provided by applicant): Circadian clocks influence nearly all aspects of mammalian life, aligning our internal physiological process to optimal times of day. Understanding the molecular circuitry keeping circadian time provides insight into how the clock drives overt rhythms, and what to fix when the circadian system is disrupted (i.e. during shift work). Circadian time coded in the rhythmic regulation of "clock gene" expression in a negative feedback loop system. Critical to this timing system is the circadian degradation of rhythmically abundant clock proteins; however these mechanisms have remained elusive. To begin elucidating these mechanisms, we developed a new functional screening approach to identify which E3 ubiquitin ligases degrade which clock proteins and screened for E3 ligases that degrade RevErb�/� proteins. Both RevErb proteins are essential for normal clock function and exhibit high amplitude abundance rhythms, but the mechanisms driving their circadian clearance are unknown. Our screen identified two novel candidate RevErb E3 ligases, Spsb4 and Siah2, and preliminary data suggest these E3s contribute directly to the rhythmic degradation of RevErb proteins. Moreover, our data suggest for the first time that the rate of circadian RevErb degradation is a determinant of circadian periodicity. Identifying the roles, mechanisms and contributions to overall clock function of Spsb4 and Siah2 is a major focus of our current research proposal. Bolstered by the fact that both hits from this screen appear to be genuine regulators of RevErb stability, the other focus of our proposal is to expand our screening efforts. Experiments proposed in Specific Aim 1 focus on elucidating the mechanisms of Siah2/Spsb4 degradation of RevErb proteins in the context of an oscillating cellular system. The experiments proposed in Specific Aim 2 delve deeper into the role of Siah2-mediated degradation of RevErb proteins in overall clock function in vivo. Experiments in Specific Aim 3 will identify candidate E3s for all of the remaining essential core clock proteins using an expanded E3 ligase cDNA screening library. Success in these aims will provide essential background and validation of our future efforts to identify protein degradation mechanisms. Overall, uncovering the novel mechanisms mediating rhythmic degradation of clock proteins will open many new avenues for treating circadian-related disorders.