Fine-Tuning the Notch Signaling Pathway via O-Glucosylation

描述(申请人提供)：Noch信号是一种进化上保守的途径，它调节多种过程，如细胞命运指定、干细胞增殖和维持、细胞死亡、隔室边界形成和皮质神经突起生长。此外，Notch信号的异常与多种人类疾病有关，包括脑血管性痴呆、癌症和影响肝脏、心脏、骨骼、眼睛和肾脏的发育障碍。尽管已经投入了大量的努力来了解在这一途径中要么全有要么全无影响的基因的功能，但关于动物如何微调信号的了解要少得多，这是一个与人类疾病发病机制和治疗学潜在高度相关的问题。这项建议的重点是描述一种新型的Notch调节器Rumi，我们已经在果蝇的无偏化学诱变筛选中发现了它。与Notch的其他调节子不同，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发挥作用，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的特定EGF重复序列中。在这项提案中，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潜在机制，验证RUMI通过改变体内Notch蛋白的糖基化模式来调节Notch信号的假设，并通过筛选RUMI的遗传修饰物来识别其他Notch“微调”基因。Rumi是一种高度保守的蛋白质，因为人类Rumi的转基因表达拯救了果蝇的Rumi突变。此外，脊椎动物的研究表明，对Notch通路的操作可以在几种疾病背景下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接口，这项拨款中提议的研究不仅将揭开动物用来调节信号的一些策略，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。 公共卫生：Notch信号的改变会导致多种人类疾病，包括癌症、心血管疾病、骨骼疾病和神经疾病。Noch信号也参与了干细胞分裂和分化的调节。在这项提案中，我们将描述葡萄糖残基如何加入到Notch蛋白中，从而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.