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

 描述(由申请人提供)：最近发现锌熔剂是各种人体生理中的关键调节事件。例如，重要的锌动员事件对皮质神经元功能、适当的免疫反应和胰岛素分泌起着至关重要的作用。锌传统上被认为是参与蛋白质结构和酶催化的静态辅因子。然而，最近的一些研究支持了这样的观点，即调控蛋白中的锌结合位点对锌可获得性的瞬时波动做出反应，并以调节关键细胞事件的方式打开和关闭。尽管锌在这些深刻的生理过程中起作用，但锌的生物化学作用还不是很清楚。关于锌流的指导机制、锌的运输途径以及特定的金属蛋白受体在信号事件中的作用，人们知之甚少。为了解释并最终干预这些通路的中断引起的神经紊乱和代谢性疾病，我们计划在分子和细胞水平上阐明锌依赖的转换事件的基本科学机制。我们将检验这样一种假设，即调节性锌熔剂 通过特定的受体介导的过程对哺乳动物的细胞周期进行有益的控制。这一假说基于多种证据，包括：(1)显示单个细胞在细胞周期不同时间点锌分布波动的数据；(2)显示锌波运动的活细胞成像；(3)显示锌与特定因素共定位的物理化学方法。我们将使用哺乳动物配子，即精子和卵子，作为一个模型细胞系统，以了解重要的锌信号通路是如何工作的。一个目标是通过检测单个细胞中锌在细胞周期中特定时间点的定位的定量变化来识别调节锌通量的分子介体和靶点。然后，我们将把这些金属可用性的时空特征与锌受体的蛋白质化学变化联系起来，锌受体是信号事件的目标。在这些研究中开发的新的化学探针和物理方法的改进和验证将大大增强我们对调控细胞决策过程的基本途径的了解。在项目结束时，我们将完成三个对生物医学界具有重要意义的创新目标。首先，我们将对无机信号事件在分子水平上的工作提供基本的新见解。其次，我们将开发方法来探索金属蛋白质组，并确定参与胚胎发生的关键蛋白如何在受精后立即改变锌的占有率。第三，我们将对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。综上所述，这些结果将阐明参与锌信号传导的蛋白质的强大作用以及细胞周期调节的机制。

 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.