In vivo Bedeutung des MAPK-Signalweg-Inhibitors SPRED - Charakterisierung der Funktion durch "knockout"-Modelle

本项目的目的是阐明SPED蛋白在体外以及在完整生物体中的多功能和复杂功能。在我们以前工作的基础上，我们打算：1.阐明SPRED2 KO小鼠应激激素产生增加的机制，这与强迫症样行为有关。我们打算研究杏仁核内突触的行为、电生理特性以及KO小鼠的解剖学特征。通过为SPRED1和-2.3建立可诱导的或组织特异性的KNOCOKOU模型，规避SPRED1和-2.3家族成员对spred蛋白的明显功能补偿和双基因敲除对胚胎的致死性。验证SPRED2与微管蛋白的蛋白质/蛋白质相互作用，通过下拉实验和随后的质谱分析进行鉴定。在分子水平上，我们想要研究SPRED2是否可能调节微管蛋白的聚合，或者是否SPRED2可能通过功能参与调节囊泡沿微管的运输。这种相互作用可能是生物学中一个基本过程的一部分，因此，可能对我们理解真核细胞的细胞内运输具有很高的相关性。动物模型提供了在整个生物体的生理背景下重新评估分子观察的可能性。

Aim of this project is to elucidate the versatile and complex functions of SPRED proteins in vitro but also in an intact organism. Based on our previous work, we intend:1. To clarify the mechanism responsible for the elevated stress hormone production in SPRED2 KO mice, which is associated with OCD-like behaviour. We intend to investigate the behaviour, electrophysiological properties of synapses in the amygdala, and anatomical characteristics of KO mice.2. To circumvent the obvious functional compensation of SPRED proteins by other family members and the embryonic lethality of double knockouts through generation of inducible or tissue-specific kncokou models for SPRED1 and -2.3. To verify the protein/protein interaction of SPRED2 with Tubulins, which was identified by pull-down assays and subsequent mass spectrometrical analyses. On the molecular level, we would like to investigate if SPRED2 might regulate Tubulin polymerisation or if SPRED2 might by functionally involved in the regulation of vesicle transport along microtubules. This interaction may be part of a fundamental process in biology and may, therefore, be of high relevance for our understanding of intracellular transport in eukaryotic cells.The animal models offer the possibilty to reassess the molecular observations in a physiological context in entire organism.