Regulated proteolysis in developmental signaling

目的I）在果蝇中模拟人类疾病的遗传异质性。的要求 >=-分泌酶在Notch膜内蛋白水解，连同丰富的 可在果蝇中分析的Notch相关分子和遗传信号输出， 促使我们探索使用转基因果蝇来模拟疾病异质性相关的 在人类早老素-1基因中有大量的突变。利用相对优势 容易和负担得起的生产转基因果蝇，我们创造了一个收集200 代表具有不同发病年龄的早老素的14种突变变体的转基因原种 人类谱系的价值观。对这些藏品的分析使我们深入了解了 β-分泌酶对不同底物的蛋白水解活性，并建立了 使用果蝇研究致病性人类突变的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 在Notch蛋白水解和细胞内运输中称为大脑（bib）的经典突变体。 Bib蛋白属于水通道蛋白家族，其转运水、离子或小分子物质。 溶质穿过生物膜。我们对围嘴的分析表明，它在 在早期内体的内吞后成熟中起作用，并导致Notch信号转导失败。 在>=-分泌酶介导的下游的特定膜运输步骤处的增殖 Notch蛋白水解。进一步研究了Bib的生化功能及其与 目前正在研究其他内体因子。目的III）分泌型的正向遗传筛选 和内体运输突变体。Notch信号传导的几种遗传调节剂，包括 我们的实验室以前的特征，最终发现影响 Notch和/或其配体在分泌和内吞区室中的运输。这些 这些发现促使我们启动了正向遗传筛选，直接寻找基因 影响Notch在高度极化上皮细胞中的细胞生物学分布 持续需要主动Notch信号传导。我们现在已经 发现了一个大的但相对特定的突变基因组， 细胞内Notch运输缺陷。这些位点的评价和进一步分析是 日至今

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.