The Ureb-1 ubiquitin ligase in neural stem cells and cancer

描述(申请人提供)：神经干细胞的快速扩增是中枢神经系统(CNS)发育的正常组成部分。然而，在小鼠发育的中期，神经前体细胞退出活跃的周期，开始分化，这个过程最初指向神经元，后来指向星形胶质细胞谱系。无节制的增殖和分化障碍是儿童和成人脑癌的特征，神经干细胞和/或未成熟的前体细胞被认为是其起源细胞。事实上，脑瘤似乎劫持了神经干细胞的动态平衡，因为脑瘤中只有一小部分细胞表现出干细胞样的特征并保留了肿瘤启动能力(脑瘤干细胞，BTSC)。在这里，我们已经确定E3泛素连接酶Ureb1是神经分化的积极调节因子和干细胞状态的抑制因子。这项提议的初步数据表明，Ureb1主要通过癌蛋白N-Myc的不稳定来发挥这些作用，N-Myc是神经干细胞扩张和填充大脑所需的一个因子，在神经肿瘤中经常被激活。我们将在这个方案中追求的模型预测，通过控制N-Myc蛋白周转，Ureb1作为抑制因子，抑制来自人类最具侵袭性的脑瘤-多形性胶质母细胞瘤(GBM)来源的BTSC的自我更新和肿瘤启动能力。在GBM中，N-Myc是控制干细胞状态的分子网络的枢纽。这项建议将利用在小鼠中可用的强大的生化和遗传工具来开发用于调节神经干细胞和来源于GBM的BTSC的动态平衡的Ureb1泛素连接酶的全面图像。首先，我们将使用一系列的生化方法来从功能上表征Ureb1蛋白的翻译后调节(磷酸化)的主要水平。我们还将利用最先进的质谱学方法来识别神经细胞中相关的含有Ureb1的蛋白质复合体。其次，我们将讨论在神经干细胞舱中携带有条件突变的Ureb1等位基因的小鼠的神经系统正常细胞和肿瘤细胞中Ureb1的功能。最后，为了直接验证Ureb1通过N-Myc对干细胞和GBM的肿瘤启动活性起刹车作用的假设，我们将操纵人GBM来源的BTSC中Ureb1和N-My的活性，并确定Ureb1基因在人类高级别胶质瘤中是否发生突变。与公共卫生相关：无节制的增殖和分化障碍是儿童和成人脑癌的特征，神经干细胞和/或未成熟的祖细胞被认为是起源细胞。N-Myc是一种参与神经干细胞状态的非常不稳定的蛋白质，在神经癌中经常被解除调控。我们已经确定了神经细胞中N-Myc的一种新的泛素连接酶，并将测试N-Myc周转缺陷是否会影响神经干细胞并引发脑部癌症。

DESCRIPTION (provided by applicant): Rapid expansion of neural stem cells is a normal component of central nervous system (CNS) development. However, at mid-gestation of mouse development, neural progenitors exit from active cycling and begin differentiation, a process that is initially directed towards the neuronal and later the astroglial lineages. Unrestrained proliferation and differentiation block are hallmarks of childhood and adult brain cancer, for which neural stem cells and/or immature progenitors have been proposed as cell of origin. Indeed, brain tumors appear to hijack the homeostasis of neural stem cells in that only a small fraction of cells within brain tumors displays stem-like features and retains tumor-initiating capacity (brain tumor stem cells, BTSC). Here we have identified the E3 ubiquitin ligase Ureb1 as a positive regulator of neural differentiation and inhibitor of the stem cell state. The preliminary data for this proposal indicate that Ureb1 exerts these effects primarily through destabilization of the oncoprotein N-Myc, a factor required for the ability of neural stem cells to expand and populate the brain and frequently activated in neural tumors. The model that we will pursue in this proposal predicts that, through the control of N-Myc protein turnover, Ureb1 acts as a restraining factor for the self- renewal and tumor initiating capacity of BTSC derived from glioblastoma multiforme (GBM), the most aggressive form of brain tumor in humans. In GBM, N-Myc is the hub of a molecular network that controls the stem cell state. This proposal will employ powerful biochemical and genetic tools available in the mouse to develop a comprehensive picture of the Ureb1 ubiquitin ligase for the regulation of the homeostasis of neural stem cells and BTSC derived from GBM. First, we will use a series of biochemical approaches to functionally characterize the major levels of post-translational regulation (phosphorylation) of the Ureb1 protein. We will also take advantage of a state-of-the-art mass spectrometry approach to identify the relevant Ureb1-containing protein complexes in neural cells. Second, we will address the function of Ureb1 in normal and tumor cells from the nervous system in mice carrying a conditionally mutant allele of Ureb1 in the neural stem cell compartment. Finally, to directly test the hypothesis that Ureb1 acts as a brake for the stem cell and tumor initiating activity of GBM through N-Myc we will manipulate the activity of Ureb1 and N-My in BTSC derived from human GBM and determine whether the Ureb1 gene is mutated in human high grade gliomas. PUBLIC HEALTH RELEVANCE: Unrestrained proliferation combined with a differentiation block are hallmarks of childhood and adult brain cancer, for which neural stem cells and/or immature progenitors have been proposed as cell of origin. N-Myc, is a very unstable protein involved in neural stem cell state and frequently deregulated in neural cancer. We have identified a new ubiquitin ligase for N-Myc in neural cells and will test whether defects in N-myc turnover affects neural stem cells and initiates cancer in the brain.