Fine-Tuning the Notch Signaling Pathway via O-Glucosylation

描述（由申请人提供）：Notch信号是一种进化上保守的信号通路，它调节多种过程，如细胞命运规范、干细胞增殖和维持、细胞死亡、室边界形成和皮质神经突生长。此外，Notch信号异常与多种人类疾病有关，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。尽管人们已经投入了大量的努力来了解在这一途径中具有全有或全无作用的基因的功能，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。本提案的重点是表征一种新的Notch调节因子rumi，我们已经在果蝇的无偏化学诱变筛选中确定了它。与Notch的其他调节因子不同，rumi的零突变表现出对温度非常敏感的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，是早老素功能的上游所必需的。生化和细胞培养研究表明，Rumi能够将葡萄糖残基添加到Notch的特异性EGF重复序列中。在本研究中，我们将结合遗传学、细胞生物学和生化分析来确定瘤胃表型温度敏感性和可逆性的机制，验证瘤胃通过改变体内Notch蛋白的糖基化模式来调节Notch信号传导的假设，并通过筛选瘤胃的遗传修饰因子来确定其他Notch“微调”基因。鲁米蛋白是一种高度保守的蛋白，人类鲁米蛋白的转基因表达可以拯救果蝇的鲁米突变。此外，脊椎动物研究表明，对Notch通路的操纵可能对几种疾病具有潜在的治疗价值，包括内耳毛细胞丢失、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，这项资助提出的研究将不仅揭示动物调节信号的一些策略，而且可能有助于改变人类疾病的结果。据我们所知，Rumi是第一个在动物中发现的蛋白质O-葡萄糖基转移酶，我们的研究也将为理解这种高度保守的修饰在后生动物生物学中的作用建立一个框架。

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.