Regulatory Roles of Zinc Fluxes in Metalloprotein Occupancy and Cell Cycle Progression

 描述（由申请人提供）：最近发现锌通量是各种人体生理学中的关键调节事件。例如，显著的锌动员事件在皮质神经元功能、适当的免疫应答和胰岛素分泌中起重要作用。传统上，锌被认为是一种参与蛋白质结构和酶催化的静态辅因子。然而，最近的一些研究已经为以下观点提供了支持：调节蛋白中的锌结合位点响应于锌可用性的瞬时波动，并且以调节关键细胞事件的方式打开和关闭。尽管锌在这些意义深远的生理学中发挥作用，但锌的生物化学作用还没有得到很好的理解。关于锌通量、锌转运途径和特定金属蛋白受体在信号事件中的作用的指导性机制知之甚少。为了解释并最终干预由这些途径中断引起的神经系统疾病和代谢疾病，我们计划在分子和细胞水平上阐明锌依赖性开关事件的基本科学机制。我们将检验调节性锌通量 通过特定的受体介导的过程对哺乳动物细胞周期进行指导性控制。这一假设基于多方面的证据，包括：（1）显示单细胞周期各个点锌分布波动的数据;（2）活细胞成像显示锌波的运动;（3）物理化学方法显示锌与特定因素的共存。我们将使用哺乳动物配子，即精子和卵子，作为模型细胞系统，以了解基本锌信号通路如何工作。一个目的是通过检测单个细胞中锌在细胞周期特定点的定位的定量变化来确定调节锌通量的分子介质和靶点。然后，我们将这些时空特征的金属的可用性与蛋白质化学的变化，锌受体的信号事件的目标。在这些研究中开发的新化学探针和物理方法的改进和验证将大大提高我们对调节细胞决策过程的基本途径的认识。在项目结束时，我们将完成对生物医学界具有重要意义的三个创新目标。首先，我们将在分子水平上提供无机信号事件工作的基本新见解。其次，我们将开发方法来探测金属蛋白质组，并确定胚胎发生中的关键蛋白质如何改变受精后立即锌占用。第三，我们将为精子获能和顶体反应中的无机信号事件提供基本的新见解。总之，这些结果将阐明一个强大的作用，蛋白质参与锌信号流和细胞周期调控的机制。

 DESCRIPTION (provided by applicant): Zinc fluxes have recently been discovered to be key regulatory events in a variety of human physiologies. For instance, significant zinc mobilization events play essential roles cortical neuron function, proper immune response, and insulin secretion. Zinc has traditionally been viewed as a static cofactor involved in protein structure and enzyme catalysis. More recently, however, a number of studies have provided support for the idea that zinc binding sites in regulatory proteins respond to transient fluctuations in zinc availability and are switched on and off in way that regulates key cellular events. Despite its rol in these profound physiologies the biochemistry of zinc action is not well understood. Little is known regarding the instructive mechanisms of zinc fluxes, zinc trafficking pathways and the role of specific metalloprotein receptors in signaling events. To interpret and eventually intervene in neurological disorders and metabolic diseases caused by disruption of such pathways, we plan to elucidate the basic scientific mechanisms of zinc-dependent switching events at the level of molecules and cells. We will test the hypothesis that regulatory zinc fluxes exert instructive control of the mammalian cell cycle through specific, receptor-mediated processes. This hypothesis is based on multiple lines of evidence, including: (1) data showing fluctuations zinc distribution at various points in the cell cycle for single cells; (2) live cell imaging demonstrating the movement of waves of zinc; and (3) physiochemical approaches showing colocalization of zinc with specific factors. We will use mammalian gametes, i.e. the sperm and egg, as a model cellular system to understand how an essential zinc signaling pathway works. One objective is to identify molecular mediators and targets of regulatory zinc fluxes by examining the quantitative changes in the localization of zinc in single cells at specifi points in the cell cycle. We will then correlate these spatio- temporal characteristics of metal availability with changes in protein chemistry of zinc receptors that are targets of the signaling events. Refinement and validation of the new chemical probes and physical methods developed in these studies will substantially enhance our knowledge of a fundamental pathway that regulates cellular decision making processes. At the project's conclusion, we will have accomplished three innovative goals of significance to the biomedical community. First, we will provide fundamental new insights into inorganic signaling events work at a molecular level. Second, we will develop methods to probe the metalloproteome and identify how key proteins involved in embryogenesis change zinc occupancy immediately following fertilization. Third, we will provide fundamental new insights into inorganic signaling events in sperm capacitation and acrosome reactions. Taken together these results will elucidate a robust role for proteins involved in zinc signaling fluxes and the mechanisms of cell cycle regulation.