COBRE: UVM MED PROJ 4: PROTEOLYTIC MODULATION OF NOTCH SIGNALING IN NEUROGENESI

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Notch信号通路由细胞表面蛋白组成，是神经系统发育过程中细胞间通讯的基础。Notch受体通过与δ蛋白结合而激活，δ蛋白可促进Notch受体内的几次连续裂解。我们最近的研究表明，Delta- notch的参与促进了Delta中类似的蛋白水解事件，这就提出了蛋白水解如何调节这一基本途径的功能的问题。Notch的切割需要一个顺序调节的膜内蛋白水解（RIP）机制，涉及ADAM金属蛋白酶和早老素（天冬氨酸蛋白酶），早老素与阿尔茨海默病的病因有关。目前，我们正在研究Notch和Delta蛋白水解在果蝇模型中的作用，通过检测Notch和Delta在培养细胞和体内的蛋白水解产物。我们的研究结果揭示了Delta蛋白的调节水解机制，它不同于Notch蛋白的调节水解机制，因为它不是顺序的，并且涉及一种不同于早老素的新型巯基敏感蛋白酶。来自Kuzbanian ADAM蛋白酶和新型巯基敏感蛋白酶的细胞相关δ切割产物分别预测了可以作用于质膜和细胞核的亚型。此外，我们已经确定了一种独特的Delta表达模式，在发育中的神经元谱系亚群中，在初始神经突接触建立的点上。总之，我们的结果指出了Delta在发育中的中枢神经系统中的一种新活动，并定义了一个进行研究的模型系统。同时，我们正在研究Notch信号对ADAM蛋白酶活性的敏感性。在这些研究中，我们已经确定了一种促进ADAM活性的有机化合物的活性。我们最近的研究结果表明，有机化合物对Notch活性的直接影响依赖于金属蛋白酶依赖性和独立活性。这些研究为我们提供了完善Notch激活机制的工具，并为研究环境形式的汞，特别是甲基汞的毒性机制提供了新的思路。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Notch signaling pathway is comprised of cell surface proteins that are fundamental to cell-cell communication in development of the nervous system. Notch receptors are activated by binding the Delta protein, which promotes several sequential cleavages in the Notch receptor. We have recently shown that Delta-Notch engagement promotes similar proteolytic events in Delta, raising the question of how proteolysis regulates the function of this fundamental pathway. Cleavage in Notch requires a sequential regulated intramembrane proteolysis (RIP) mechanism involving an ADAM metalloprotease and Presenilin, the aspartyl protease involved in the etiology of Alzheimer's disease. Surrently we are investigating the role of proteolysis of Notch and Delta in a Drosophila (fruit fly) model by examining the proteolytic products of Notch and Delta in both cultured cells and in vivo. Our results have uncovered a mechanism of regulated proteolysis of Delta that is distinct from that of Notch in that it is not sequential and involves a novel thiol-sensitive protease that is distinct from Presenilin. Cell associated Delta cleavage products derived from the Kuzbanian ADAM protease and the novel thiol-sensitive protease predict isoforms that can act at the plasma membrane and in the nucleus, respectively. Furthermore, we have identified a unique pattern of Delta expression in subsets of developing neuronal lineages at the point in which initial neurite contacts are established. Altogether, our results point to a novel activity for Delta in the developing central nervous system and define a model system in which to carry out the investigation. Also we are studying the sensitivity of Notch signaling to ADAM protease activity. In these studies we have identified an activity of organomercurial compounds in promoting ADAM activity. Our recent findings demonstrate a direct effect of organomercurials on Notch activity, which relies on both metalloprotease-dependent and independent activities. These studies give us the tools to refine the mechanism of Notch activation, as well as provide new lines of inquiry into the toxic mechanisms of environmental forms of mercury, notably methylmercury.