Regulated proteolysis in developmental signaling

项目1：Notch受体的运输和翻译后修饰。我们已经研究了Notch受体蛋白的运输和翻译后修饰的作用几年了。Notch信号通路控制许多不同细胞类型和组织的分化，并且该通路的故障已被证明会导致人类T细胞白血病和其他疾病。最近，我们已经确定了一个突变体，改变了内吞内化的Notch，导致Notch积累在扩大的内体囊泡。该突变影响了编码通道相关蛋白的基因，我们目前正在研究这种蛋白的分子和生化特性。初步数据表明，这类通道蛋白的化学抑制剂损害果蝇培养细胞中Notch受体的蛋白水解激活，这表明新因子在信号通路的这一步骤中的作用。突变果蝇组织克隆的细胞生物学研究以及标记的Notch途径组分的遗传和生化分析正在用于研究这种新的通道相关蛋白在信号通路中的功能。项目2：果蝇中与哺乳动物β-淀粉样蛋白结合蛋白相关的基因家族的分析。近年来的大量研究发现了一种称为γ-分泌酶的多蛋白复合物，其在阿尔茨海默病的Notch信号传导和淀粉样肽产生中具有保守的蛋白水解功能。Notch和淀粉样蛋白前体蛋白（APP）（淀粉样蛋白肽的来源）都是1型单程整合膜蛋白，是γ-分泌酶介导的膜内蛋白水解的底物。果蝇基因组编码三种与已知结合淀粉样蛋白的哺乳动物蛋白相关的蛋白质，其中一种果蝇蛋白质被认为参与Notch信号相关的发育过程。这些观察结果促使我们开始对该基因家族进行遗传和分子表征，目的是阐明其在Notch生物合成、运输、活化、降解或受体代谢的其他方面的潜在功能。RNA干扰（RNAi），生物化学和共聚焦显微镜的方法在培养细胞和转基因苍蝇的组合正在利用这个项目。

Project 1: Trafficking and posttranslational modification of the Notch receptor. We have been studying the role of trafficking and posttranslational modification of the Notch receptor protein for several years. The Notch signaling pathway controls the differentiation of many different cell types and tissues, and malfunction of this pathway has been shown to cause T-cell leukemia and other diseases in humans. Recently we have identified a mutant which alters the endocytic internalization of Notch, causing Notch to accumulate in enlarged endosomal vesicles. The mutation affects a gene encoding a channel-related protein, and we are currently investigating the molecular and biochemical properties of this protein. Preliminary data indicate that chemical inhibitors of this class of channel proteins impair the proteolytic activation of the Notch receptor in Drosophila cultured cells, suggesting a role for the new factor in this step of the signaling pathway. Cell biological studies in mutant Drosophila tissue clones together with genetic and biochemical analyses of tagged Notch pathway components are being used to investigate the function of this new channel-related protein in the signaling pathway.- Project 2: Analysis of a gene family in Drosophila related to mammalian beta-amyloid binding protein. Numerous studies in recent years have uncovered a multiprotein complex termed gamma-secretase that has a conserved proteolytic function in both Notch signaling and amyloid peptide production in Alzheimer's disease. Both Notch and Amyloid Precursor Protein (APP), from which amyloid peptide is derived, are type 1 single-pass integral membrane proteins that are substrates for gamma-secretase-mediated intramembrane proteolysis. The Drosophila genome encodes three proteins related to a mammalian protein known to bind amyloid, and one of these fly proteins is thought to participate in Notch signaling-related developmental processes. These observations prompted us to initiate a genetic and molecular characterization of this gene family with the aim of elucidating its potential function(s) in Notch biosynthesis, trafficking, activation, degradation, or other aspects of receptor metabolism. A combination of RNA interference (RNAi), biochemical, and confocal microscopy approaches in cultured cells and transgenic flies are being utilized in this project.