Role of Protein Arginylation in Cardiovascular Development

项目概述：本提案将研究新发现的翻译后修饰- n端蛋白精氨酸化-在心血管发育中的作用。PI先前发表的文章表明，蛋白精氨酸化对心血管发育至关重要，敲除蛋白精氨酸化酶ATE1会导致小鼠胚胎致死和严重的心血管表型，这与人类最常见的先天性心脏缺陷非常相似。本研究旨在通过以下三个具体目标，全面了解蛋白精氨酸化在心血管发育中的作用，并深入了解先天性心脏病的可能机制和治疗方法：(1)通过对培养胚胎细胞的活体观察、免疫组织化学、原位杂交和微阵列分析，验证精氨酸化调节细胞迁移和规范并可能影响心脏发育过程中凋亡率的假设；(2)通过条件小鼠Ate1基因敲除，鉴定导致Ate1 -/-发育表型的细胞系，并验证这种表型至少部分是由于神经嵴、内皮细胞系和心肌细胞的变化所导致的假设；(3)通过生化分析和质谱分析，鉴定体内心血管组织中精氨酸化的蛋白，并确定Ate1-/-心血管表型的可能分子机制。综上所述，所提出的实验将允许确定由蛋白质精氨酸化控制的心血管发育的特定途径，并在基因表达和蛋白质水平上深入了解这种控制的分子机制。

DESCRIPTION (provided by applicant): Project summary: This proposal will investigate the role of a newly rediscovered posttranslational modification-N-terminal protein arginylation-in cardiovascular development. Prior publications by the PI demonstrate that protein arginylation is critical for cardiovascular development and that knockout of the protein arginylation enzyme ATE1 results in embryonic lethality and severe cardiovascular phenotypes in mice that closely resemble the most commonly occurring congenital heart defects in humans. The proposed research is aimed to gain a general understanding of the role of protein arginylation in cardiovascular development and gain insights into possible mechanisms and ways of treatment of the congenital heart diseases, through the following three specific aims: (1) To test the hypothesis that arginylation regulates cell migration and specification and possibly affects the rates of apopotosis during heart development, using live observations of cultured embryonic cells, immunohistochemistry, insitu hybridization, and microarray profiling; (2) To identify cell lineages responsible for the Ate1 -/- developmental phenotype and to test the hypothesis that this phenotype is due at least in part to changes in cells of the neural creast and endothelial lineages and cardiac myocytes, using conditional mouse ATE1 knockouts; and (3) To identify proteins arginylated in vivo in cardiovascular tissues and to define the possible molecular mechanisms of the Ate1-/- cardiovascular phenotype, using biochemical analysis and mass spectrometry. Taken together, the proposed experiments will allow to identify the specific pathways in cardiovascular development controlled by protein arginylation and to gain insights into the molecular mechanisms of this control at the gene expression and protein level. Relevance to public health: Ate1-/- cardiovascular phenotypes resemble commonly occurring human congenital heart diseases, including ventricular and atrial septal defects (VSD and ASD), persistent truncus artheriosis (PTA), and DiGeorge syndrome. The proposed research is aimed to address the previously unexplored mechanisms of these diseases and provide insights into the new possibilities of their treatment.