Structural, physiological and pathogenic features of APP/APLPs E1 domain

APP/APLPs E1结构域的结构、生理和致病特征

• 批准号: 173182206
• 负责人: Professor Dr. Stefan Kins
• 金额: --
• 依托单位: Fachgebiet Humanbiologie und Humangenetik
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/173182206?language=en
• 关键词: Structural; physiological; pathogenic; features; APP

Deletion of the APP family members leads to a decreased neurotransmission at the neuromuscular junction, possibly caused by a loss of fianction of APP family proteins in neuronal cell adhesion. We assume that APP/APLPs dependent cell adhesion is mediated by direct trans- interaction of the E1 domain of APP/APLPs. To test this hypothesis, we intend to purify the El domain and the complete extracellular domain of APP/APLPs, using His-tagged mutant constructs. The purified proteins will be subjected to crystallization and X-ray structural analysis as well as biophysical characterizafion like Isothermal Titration Calorimetry and Static Light Scattering to determine their dimerization properties. Secondly, we will use an in vitro trans-dimerization assay to purify and determine modulators of homotypic APLPl and APLP2 trans-interaction. Similarly, we will screen for modulators of APP/APLPs heterotypic trans-dimerization. The novel putative APP/APLPs trans-dimerization modifiers will be tested in cell based clustering assays and primary neuronal cultures. Thirdly, we will determine the subcellular localization of APP/APLPs at the synapse and at extra-synapfic sites at the plasma membrane, using high-resolution light microscopy. Finally, we will investigate if the extracellular domain of APP/APLPs is sufficient to induce post- or presynaptic differentiation and will determine the sequence of underlying molecular and cellular events by life cell imaging. All together these analyses will help to understand the function of homo- and heterotypic trans-interaction of the APP gene family at the synapse.

APP 家族成员的缺失会导致神经肌肉接头处的神经传递减少，这可能是由于 APP 家族蛋白在神经元细胞粘附中的功能丧失所致。我们假设 APP/APLP 依赖性细胞粘附是由 APP/APLP 的 E1 结构域的直接反式相互作用介导的。为了检验这一假设，我们打算使用 His 标记的突变构建体纯化 APP/APLP 的 El 结构域和完整的胞外结构域。纯化的蛋白质将进行结晶和 X 射线结构分析以及生物物理表征，如等温滴定量热法和静态光散射，以确定其二聚特性。其次，我们将使用体外反式二聚化测定来纯化和确定同型APLP1和APLP2反式相互作用的调节剂。同样，我们将筛选 APP/APLP 异型反二聚化的调节剂。新型假定的 APP/APLP 反式二聚修饰剂将在基于细胞的聚类分析和原代神经元培养物中进行测试。第三，我们将使用高分辨率光学显微镜确定 APP/APLP 在突触和质膜突触外位点的亚细胞定位。最后，我们将研究 APP/APLP 的胞外结构域是否足以诱导突触后或突触前分化，并将通过生命细胞成像确定潜在分子和细胞事件的序列。总之，这些分析将有助于理解 APP 基因家族在突触处的同型和异型反式相互作用的功能。