Regulated proteolysis in developmental signaling

Notch受体的转运和翻译后修饰。多年来，我们一直在研究Notch受体蛋白的转运和翻译后修饰的作用。Notch信号通路控制着许多不同细胞类型和组织的分化，该通路的功能障碍已被证明可导致人类t细胞白血病和其他疾病。最近，我们发现了一个突变，它改变了Notch的内吞内化，导致Notch积聚在增大的内体囊泡中。该突变影响编码通道相关蛋白的基因，我们目前正在研究该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了果蝇培养细胞中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中起作用。突变果蝇组织克隆的细胞生物学研究以及标记Notch通路组分的遗传和生化分析正在被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，它在阿尔茨海默病的Notch信号传导和淀粉样肽的产生中都具有保守的蛋白水解功能。Notch和淀粉样蛋白前体蛋白（APP）都是1型单道完整膜蛋白，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蛋白质，这些蛋白质与一种已知与淀粉样蛋白结合的哺乳动物蛋白质有关，其中一种果蝇蛋白质被认为参与了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、激活、降解或受体代谢的其他方面的潜在功能。RNA干扰（RNAi）、生化和共聚焦显微镜方法的结合在培养细胞和转基因苍蝇中被用于这个项目。-项目3：果蝇人类疾病发病变异性的转基因建模。γ -分泌酶的核心成分之一天冬氨酸蛋白酶早老素的突变可导致常染色体显性早发性阿尔茨海默病。然而，在受影响的家庭中，发病年龄是高度可变的，并且可能由早老素本身的原发病变以及其他遗传和/或环境因素决定。为了测试发病年龄的差异是否可以在一个定义良好的遗传模式生物中重现，我们将14种疾病相关的早老素突变体引入转基因果蝇，并评估它们在缺乏内源性早老素活性的动物中的相对功能程度。到目前为止获得的结果表明，在转基因果蝇模型中，每种早老素突变的发病年龄与功能损伤的相对程度有很好的相关性，这些发现表明，发病年龄主要取决于每种突变对早老素活性的有害影响，其他环境或遗传因素不太可能在该病的发病年龄中起主要作用。并且果蝇可以成功地用于模拟人类遗传疾病的分级特征。

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.- Project 3: Transgenic modeling of human disease onset variability in Drosophila. Mutations in one of the core components of gamma-secretase, the aspartyl protease Presenilin, cause autosomal dominant, early-onset Alzheimer's disease. However, age of onset is highly variable in affected families, and might be determined by the primary lesions in Presenilin itself as well as additional genetic and/or environmental factors. To test whether the differences in age of onset can be recapitulated in a well-defined genetic model organism, we have introduced a select panel of 14 disease-associated Presenilin mutants into transgenic Drosophila and are assessing their relative degree of function in animals lacking endogenous Presenilin activity. Results obtained so far indicate that age of onset for each Presenilin mutation correlates well with relative degree of functional impairment in the transgenic fly model, These findings indicate that age of onset is primarily determined by the deleterious effects of each mutations on Presenilin activity, that other environmental or genetic factors are unlikely to play a major role in age of onset in this disease, and that Drosophila can be used successfully to model graded characteristics of human genetic diseases.