Fine-Tuning the Notch Signaling Pathway via O-Glucosylation

描述（由申请人提供）：Notch信号传导是一种进化上保守的途径，其调节细胞命运特化、干细胞增殖和维持、细胞死亡、隔室边界形成和皮质神经突生长等多种过程。此外，异常Notch信号传导涉及多种人类疾病，包括脑血管痴呆、癌症和影响肝脏、心脏、骨骼、眼睛和肾脏的发育障碍。虽然已经投入了大量的努力来了解在这一途径中具有全或无效应的基因的功能，但对动物如何微调信号传导知之甚少，这是一个与人类疾病发病机制和治疗方法潜在高度相关的问题。这个建议的重点是一个新的Notch调节器鲁米，我们已经确定在果蝇的无偏见的化学诱变筛选的表征。与Notch的其他调节因子不同，rumi的无效突变表现出显著的温度敏感性Notch功能丧失表型。体内分析表明Rumi在信号接收细胞的内质网中起作用，并且是早老素功能上游所需的。生物化学和细胞培养研究表明，Rumi能够将葡萄糖残基添加到Notch的特定EGF重复序列中。在这个建议中，我们将使用遗传学，细胞生物学和生化分析相结合，以确定潜在的温度敏感性和可逆性的鲁米表型的机制，以测试的假设，鲁米调节Notch信号通过改变糖基化模式的Notch蛋白在体内，并确定其他Notch的“微调”基因通过筛选遗传修饰的鲁米。Rumi是一种高度保守的蛋白质，因为人Rumi的转基因表达挽救了果蝇中的Rumi突变。此外，脊椎动物研究表明，Notch途径的操纵在几种疾病背景下可能具有潜在的治疗价值，包括内耳毛细胞损失、肌肉损伤和脱髓鞘。因此，我们希望通过揭示细胞生物学和发育的界面，这项资助中提出的研究不仅将揭示动物用于调节信号传导的一些策略，而且还可能有助于改变人类疾病结果的努力。由于据我们所知，Rumi是第一个在动物中鉴定的蛋白质O-葡糖基转移酶，我们的研究也将建立一个框架，以了解这种高度保守的修饰在后生动物生物学中的作用。 公共卫生部门：Notch信号传导的改变导致多种人类疾病，包括癌症、心血管、骨骼和神经系统疾病。Notch信号也参与干细胞分裂和分化的调节。在这个提议中，我们将描述如何添加葡萄糖残基的Notch蛋白微调这一重要途径介导的信号。

DESCRIPTION (provided by applicant): Notch signaling is an evolutionarily conserved pathway that regulates processes as diverse as cell fate specification, stem cell proliferation and maintenance, cell death, compartment boundary formation and cortical neurite outgrowth. Also, aberrant Notch signaling is involved in a variety of human diseases including cerebrovascular dementia, cancer and developmental disorders affecting liver, heart, skeleton, eye, and kidney. Although a lot of effort has been devoted to understanding the function of the genes with all-or-none effects in this pathway, much less is known about how animals fine-tune signaling, an issue of potentially high relevance to human disease pathogenesis and therapeutics. The focus of this proposal is on the characterization of a novel Notch regulator rumi, which we have identified in an unbiased chemical mutagenesis screen in Drosophila. Unlike other regulators of Notch, null mutations of rumi exhibit a dramatically temperature-sensitive Notch loss-of- function phenotype. In vivo analysis indicates that Rumi functions in the endoplasmic reticulum of the signal-receiving cell and is required upstream of the Presenilin function. Biochemical and cell culture studies have shown that Rumi is able to add glucose residues to specific EGF repeats of Notch. In this proposal we will use a combination of genetic, cell biological and biochemical analyses to identify the mechanism underlying the temperature-sensitivity and reversibility of the rumi phenotype, to test the hypothesis that Rumi regulates Notch signaling via altering the glycosylation pattern of the Notch protein in vivo, and to identify other Notch "fine-tuning" genes by screening for genetic modifiers of rumi. Rumi is a highly conserved protein, as transgenic expression of human Rumi rescues rumi mutations in flies. Moreover, vertebrate studies have shown that manipulation of the Notch pathway can be of potential therapeutic value in several disease contexts, including inner ear hair cell loss, muscle injury and demyelination. Therefore, our hope is that by shedding light on the interface of cell biology and development, the research proposed in this grant will not only unravel some of the strategies used by animals to regulate signaling, but might also contribute to efforts aimed at altering the outcome of human diseases. Since to our knowledge Rumi is the first protein O- glucosyltransferase identified in animals, our studies will also establish a framework for understanding the role of this highly conserved modification in metazoan biology. PUBLIC HEALTH REVELANCE: Alterations in Notch signaling causes a variety of human diseases including cancer, cardiovascular, skeletal and neurological disorders. Notch signaling is also involved in the regulation of stem cell division and differentiation. In this proposal we will characterize how addition of glucose residues to the Notch protein fine-tunes signaling mediated by this important pathway.