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

描述（由申请人提供）：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，在小鼠发育的妊娠中期，神经祖细胞退出活跃循环并开始分化，这一过程最初指向神经元谱系，后来指向星形胶质谱系。无节制的增殖和分化阻断是儿童和成人脑癌的特征，神经干细胞和/或未成熟祖细胞被认为是其起源细胞。事实上，脑肿瘤似乎劫持了神经干细胞的体内平衡，因为脑肿瘤中只有一小部分细胞表现出类似干细胞的特征，并保留了肿瘤启动能力（脑肿瘤干细胞，BTSC）。在这里，我们已经确定了E3泛素连接酶Ureb1作为神经分化的积极调节因子和干细胞状态的抑制剂。该提案的初步数据表明，Ureb1主要通过破坏癌蛋白N-Myc的稳定来发挥这些作用，N-Myc是神经干细胞扩张和填充大脑的能力所必需的因子，在神经肿瘤中经常被激活。我们将在本提案中追求的模型预测，通过控制N-Myc蛋白的转换，Ureb1作为一个抑制因子，抑制来自多形性胶质母细胞瘤（GBM）的BTSC的自我更新和肿瘤启动能力，GBM是人类最具侵袭性的脑肿瘤。在GBM中，N-Myc是控制干细胞状态的分子网络的中心。本研究将利用强大的小鼠生物化学和遗传学工具，全面了解ub1泛素连接酶对GBM衍生的神经干细胞和BTSC稳态调节的作用。首先，我们将使用一系列生化方法从功能上表征Ureb1蛋白翻译后调控（磷酸化）的主要水平。我们还将利用最先进的质谱方法来鉴定神经细胞中相关的含ureb1的蛋白质复合物。其次，我们将研究Ureb1在神经干细胞区携带Ureb1条件突变等位基因的小鼠神经系统正常细胞和肿瘤细胞中的功能。最后，为了直接验证Ureb1通过N-Myc抑制GBM干细胞和肿瘤启动活性的假设，我们将操纵人类GBM BTSC中Ureb1和N-My的活性，并确定Ureb1基因是否在人类高级别胶质瘤中发生突变。公共卫生相关性：无节制的增殖结合分化阻断是儿童和成人脑癌的标志，神经干细胞和/或未成熟祖细胞被认为是其起源细胞。N-Myc是一种非常不稳定的蛋白，参与神经干细胞状态，在神经肿瘤中经常失控。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转换的缺陷是否会影响神经干细胞并引发脑癌。

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.