Trafficking and Proteolysis of Notch and Other Gamma-Secretase Substrates

描述（由申请人提供）：许多完整的膜蛋白已被确定为膜内乙酰基蛋白酶复合物的切割底物，称为？分泌酶这些？分泌酶的底物包括信号通路的受体、粘附分子和其他定位于细胞表面的因子，它们的活性受β-淀粉样蛋白调节。分泌酶介导的裂解。了解分子的细节？？分泌酶底物生物学是重要的，因为许多底物在正常细胞生理学和人类疾病中起重要作用。例如，Notch受体，一种很好表征的？作为分泌酶底物，在动物和人体组织发育过程中对适当的细胞命运特化和细胞分化至关重要。Notch通路在人类免疫系统中的不适当激活导致T细胞白血病，并且Notch活性失调与其他癌症和发育疾病有关。此外，淀粉样前体蛋白（APP）的加工？分泌酶直接促进人脑组织中分泌的淀粉样蛋白-2肽的产生，这是阿尔茨海默病发病机制中的关键事件。一个关键的特点？？分泌酶底物切割是其通过配体结合、胞外域去除和其它调节过程的严格控制。越来越多的数据表明，切割也依赖于细胞内膜运输？分泌酶底物及其在特定运输区室中的分选。在Notch和APP的情况下，？？分泌酶介导的切割与底物从细胞表面的内吞作用及其进入内体有关。我们已经发现，早期内体区室的适当生物发生需要水通道蛋白的功能，定义了一个专门的内体途径，以有效的？分泌酶依赖的Notch运输和信号传导。在具体目标1中，我们建议进一步表征这种水通道蛋白在内体生物发生和Notch运输中的作用。这些研究将阐明细胞生物学方面的？？分泌酶底物运输及其与？？分泌酶底物裂解、再循环和降解。在具体目标2中，我们建议进行正向遗传筛选，以寻找参与Notch和其他？分泌酶底物。该筛选的初步结果已经被证明是非常有前途的，包括Notch细胞内运输的各个步骤所需的许多潜在的新基因的回收。目前正在对这些新基因进行进一步的分析和分子克隆。这个项目的主要目标是促进我们对？？分泌酶底物运输及其与肿瘤发生和神经退行性疾病中分泌酶依赖过程的相关性。公共卫生相关性：由？？切割的蛋白质分泌酶，包括Notch受体和淀粉样前体蛋白，对于发育信号传导和神经元功能是重要的，并且它们的失调有助于癌症和神经退行性疾病。这项研究计划旨在确定细胞因子控制生产，运输和蛋白水解加工？分泌酶的目标。这些研究可能会导致识别新的蛋白质和细胞内途径，这些蛋白质和细胞内途径可能是癌症和神经退行性疾病的治疗靶点。

DESCRIPTION (provided by applicant): Many integral membrane proteins have been identified as cleavage substrates for the intramembrane aspartyl protease complex termed ??secretase. These ??secretase substrates include receptors for signaling pathways, adhesion molecules, and other factors that are localized at the cell surface and whose activities are regulated by ??secretase-mediated cleavage. Understanding the molecular details of ??secretase substrate biology is important due to the prominent role that many substrates play in normal cellular physiology and in human disease. For example, the Notch receptor, a well- characterized ??secretase substrate, is essential for proper cell-fate specification and cellular differentiation during the development of animal and human tissues. Inappropriate activation of the Notch pathway in the human immune system causes T-cell leukemia and dysregulated Notch activity has been implicated in other cancers and developmental diseases. Furthermore, processing of the Amyloid Precursor Protein (APP) by ??secretase contributes directly to the production of secreted amyloid-2 peptide in human brain tissue, a crucial event in the pathogenesis of Alzheimer's disease. A key feature of ??secretase substrate cleavage is its tight control by ligand binding, ectodomain removal, and other regulatory processes. A growing body of data indicates that cleavage also depends upon intracellular membrane trafficking of ??secretase substrates and their sorting within specific trafficking compartments. In the case of both Notch and APP, ??secretase-mediated cleavage is associated with endocytosis of the substrate from the cell surface and its entry into endosomes. We have found that proper biogenesis of the early endosome compartment requires the function of an aquaporin channel protein, defining a specialized endosome route for efficient ??secretase-dependent Notch trafficking and signaling. In Specific Aim 1, we propose to characterize further the role of this aquaporin in endosome biogenesis and Notch trafficking. These studies will shed light on the cell biological aspects of ?? secretase substrate trafficking and their relationship to ??secretase substrate cleavage, recycling, and degradation. In Specific Aim 2, we propose to conduct a forward genetic screen for new factors involved in the intracellular membrane trafficking of Notch and other ??secretase substrates. Preliminary results from this screen have already proven to be very promising, including the recovery of many potentially new genes needed for various steps of Notch intracellular trafficking. Further analysis and molecular cloning of several of these new genes is now underway. The main goal of this project is to advance our understanding of ??secretase substrate trafficking and its relevance to secretase- dependent processes in tumorigenesis and neurodegenerative disease. PUBLIC HEALTH RELEVANCE: Proteins cleaved by ??secretase, including the Notch receptor and amyloid precursor protein, are important for developmental signaling and neuronal function, and their dysregulation contributes to cancer and neurodegenerative disease. This research proposal seeks to identify cellular factors that control the production, trafficking, and proteolytic processing of ?? secretase targets. These studies might lead to the identification of new proteins and intracellular pathways that could be therapeutically targeted in cancer and neurodegenerative disorders.