Degradation Mechanisms of Mammalian Circadian Clock Proteins

描述（由申请人提供）：生物钟影响哺乳动物生活的几乎所有方面，使我们的内部生理过程与一天中的最佳时间相一致。了解保持昼夜节律时间的分子电路可以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破坏时（即在轮班工作期间）需要修复什么。负反馈环系统中生物钟基因表达节律调控的昼夜节律时间编码。这个计时系统的关键是节律丰富的时钟蛋白的昼夜降解;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鉴定哪些E3泛素连接酶降解哪些时钟蛋白，并筛选降解RevErb β/β蛋白的E3连接酶。这两种RevErb蛋白对正常的生物钟功能都是必不可少的，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。 我们的筛选鉴定了两种新的候选RevErb E3连接酶，Spsb 4和Siah 2，初步数据表明这些E3直接有助于RevErb蛋白的节律性降解。此外，我们的数据首次表明，昼夜RevErb降解率是昼夜周期性的决定因素。确定Spsb 4和Siah 2的角色，机制和对整体时钟功能的贡献是我们目前研究计划的主要重点。由于这个屏幕上的两个点击似乎是RevErb稳定性的真正监管者，我们建议的另一个重点是扩大我们的筛选工作。 具体目标1中提出的实验集中于阐明在振荡细胞系统的背景下Siah 2/Spsb 4降解RevErb蛋白的机制。Specific Aim 2中提出的实验深入研究了Siah 2介导的RevErb蛋白降解在体内整体时钟功能中的作用。具体目标3中的实验将使用扩展的E3连接酶cDNA筛选文库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。总的来说，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.