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，调节细胞命运规范、干细胞增殖和维持、细胞死亡、区室边界形成和皮质神经突生长等多种过程。此外，异常的 Notch 信号传导还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。尽管人们付出了大量努力来了解该途径中具有全或无影响的基因的功能，但对于动物如何微调信号传导却知之甚少，而这一问题与人类疾病发病机制和治疗可能高度相关。该提案的重点是一种新型Notch调节器rumi的表征，我们在果蝇的无偏化学诱变筛选中发现了它。与其他Notch调节因子不同，rumi的无效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发挥作用，并且是早老素功能上游所必需的。生化和细胞培养研究表明，Rumi 能够将葡萄糖残基添加到 Notch 的特定 EGF 重复序列中。在本提案中，我们将结合遗传、细胞生物学和生化分析来确定rumi表型的温度敏感性和可逆性的机制，检验Rumi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rumi的遗传修饰剂来鉴定其他Notch“微调”基因。 Rumi 是一种高度保守的蛋白质，人类 Rumi 的转基因表达可以挽救果蝇中的 Rumi 突变。此外，脊椎动物研究表明，Notch 通路的操纵在多种疾病中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，我们希望通过阐明细胞生物学和发育的界面，本次资助中提出的研究不仅将阐明动物用于调节信号传导的一些策略，而且还可能有助于旨在改变人类疾病结果的努力。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，我们的研究还将建立一个框架来理解这种高度保守的修饰在后生动物生物学中的作用。 公共卫生启示：Notch 信号传导的改变会导致多种人类疾病，包括癌症、心血管、骨骼和神经系统疾病。 Notch信号还参与干细胞分裂和分化的调节。在本提案中，我们将描述向 Notch 蛋白添加葡萄糖残基如何微调这一重要途径介导的信号传导。

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.