Direct and Quantitative Proteomic Approaches in Xenopus

描述（由申请人提供）： 本研究的目的是开发和优化非洲爪蟾蛋白质组学研究的技术和试剂。具体而言，我们提出了一系列的可行性研究的基础上的三种蛋白质，T-盒转录因子TBX 5，锌指转录因子CASZ 1，和非受体蛋白磷酸酶SHP 2。我们专注于这些蛋白质，基于它们在序列，表达和功能方面的进化保守性，它们的细胞定位，以及观察到这些蛋白质是致病的，或与人类疾病状态有关：患有Holt-Oram综合征（HOS）（一种也影响心脏和四肢的疾病）的患者与Tbx 5中的错义突变有关，全基因组关联研究暗示Casz 1在高血压和高血压中的作用，患有努南综合征（一种也影响心脏和肢体发育的疾病）的患者通常与Shp-2中的错义突变相关。尽管TBX 5、CASZ 1和SHP-2在发育和人类疾病中起着至关重要的作用，但这些蛋白在体内发挥作用的分子机制仍有待建立。为了解决这些问题，并建立一套通用的技术，用于定向蛋白质组学方法在非洲爪蟾，我们建议1）建立一个二元转基因系统在非洲爪蟾蛋白质和蛋白质-DNA复合物的分离和2）建立技术，试剂和协议，定向和定量蛋白质组学方法在非洲爪蟾。

DESCRIPTION (provided by applicant): ABSTRACT The goal of this proposal is to develop and optimize the technologies and reagents for use of directed and quantitative proteomic approaches in Xenopus. Specifically, we propose a series of feasibility studies based on three proteins, the T-box transcription factors TBX5, the zinc finger transcription factor CASZ1, and the non-receptor protein phosphatase SHP2. We have focused on these proteins based on their evolutionary conservation in sequence, expression, and function, their cellular localization, and the observation that these proteins are causative, or have association with human disease states: patients with Holt-Oram syndrome (HOS), a disease which also affects the heart and limbs, is associated with mis-sense mutations in Tbx5, genome wide association studies have implied a role for Casz1 in hypertension and high blood pressure, and patients with Noonan syndrome, a disease that also affects heart and limb development, frequently is associated with mis-sense mutations in Shp-2. Despite the crucial role for TBX5, CASZ1 and SHP-2 in development and human disease, the molecular mechanisms by which these proteins act in vivo remains to be established. To address these issues and to establish a set of general technologies for the use of directed proteomics approaches in Xenopus we propose to 1) establish a binary transgenic system in Xenopus for the isolation of protein and protein-DNA complexes and 2) establish the technologies, reagents and protocols for directed and quantitative proteomic approaches in Xenopus.