Regulated proteolysis in developmental signaling

目的1)在果蝇中建立人类疾病遗传异质性模型。Notch膜内蛋白水解对>=-分泌酶的需求，以及果蝇中可以分析的丰富的Notch相关分子和遗传信号输出，促使我们探索利用转基因果蝇来模拟与人类早老素-1基因大量突变相关的疾病异质性。利用生产转基因果蝇相对容易和负担得起的优势，我们创建了200个转基因种群，代表14个早老素突变体，它们在人类谱系中具有不同的发病年龄值。对这一集合的分析揭示了>=-secretase对不同底物的保守蛋白水解活性，并建立了利用果蝇研究致病性人类突变等位基因特异性特征的有效性。目的II)内体生物发生和Notch激活。我们正在研究一种称为big brain （bib）的经典突变体在Notch蛋白水解和细胞内运输中的功能。Bib蛋白属于水通道蛋白通道家族，它通过生物膜运输水、离子或小溶质。我们对Bib的分析表明，它在早期核内体的内吞后成熟中起重要作用，并在>=-分泌酶介导的Notch蛋白水解的下游特定膜运输步骤中导致Notch信号传播失败。目前正在进一步研究Bib的生化功能及其与其他内体因子的关系。目标三)对分泌和内体转运突变体进行遗传筛选。Notch信号的几个遗传调节剂，包括我们实验室之前描述的那些，最终被发现影响Notch和/或其配体在分泌和内吞区室中的运输。这些发现促使我们启动正向遗传筛选，直接寻找影响Notch在影像盘高度极化上皮细胞中细胞生物学分布的基因，这些细胞生物学分布持续需要活跃的Notch信号。我们现在已经确定了一个大而相对特定的突变基因组，在细胞内Notch运输中显示出特定的缺陷。目前正在对这些基因座进行评价和进一步分析。

Aim I) Modeling human disease genetic heterogeneity in Drosophila. The requirement for >=-secretase in Notch intramembrane proteolysis, together with the wealth of Notch-related molecular and genetic signal outputs that can be analyzed in Drosophila, prompted us to explore the use of transgenic flies to model disease heterogeneity associated with a large set of mutations in the human Presenilin-1 gene. Taking advantage of the relative ease and affordability of producing transgenic Drosophila, we created a collection of 200 transgenic stocks representing 14 mutant variants of Presenilin having different age of onset values in human pedigrees. Analysis of this collection yielded insights into the conserved proteolytic activity of >=-secretase towards different substrates, and established the validity of using Drosophila to study allele-specific features of pathogenic human mutations. Aim II) Endosomal biogenesis and Notch activation. We are investigating the function of a classical mutant termed big brain (bib) in Notch proteolysis and intracellular trafficking. The Bib protein belongs to the aquaporin channel family, which transport water, ions, or small solutes across biological membranes. Our analysis of Bib revealed that it plays an important role in post-endocytic maturation of early endosomes, and causes a failure in Notch signal propagation at a specific membrane trafficking step downstream of >=-secretase-mediated Notch proteolysis. Further studies on the biochemical function of Bib and its relationship to other endosomal factors are currently underway. Aim III) Forward genetic screen for secretory and endosomal trafficking mutants. Several genetic modulators of Notch signaling, including ones characterized previously by our laboratory, were ultimately found to affect the trafficking of Notch and/or its ligands in the secretory and endocytic compartments. These findings prompted us to initiate forward genetic screens to search directly for genes affecting the cell biological distribution of Notch in the highly polarized epithelial cells of the imaginal discs, which continuously require active Notch signaling. We have now identified a large yet relatively specific group of mutant genes that display particular defects in intracellular Notch trafficking. Evaluation and further analysis of these loci are ongoing.