Role of Protein Arginylation in Cardiovascular Development

描述(由申请人提供)：项目摘要：本提案将调查新发现的翻译后修饰-N-末端蛋白精氨酸化-在心血管发育中的作用。PI以前发表的文章表明，蛋白质精氨酸化对心血管发育至关重要，蛋白质精氨酸化酶ATE1的敲除会导致小鼠胚胎死亡和严重的心血管表型，这与人类最常见的先天性心脏病非常相似。本研究旨在通过以下三个具体目标，对蛋白质精氨酸化在心血管发育中的作用以及对先天性心脏病可能的机制和治疗方法有一个全面的了解：(1)通过对培养的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指定并可能影响心脏发育过程中细胞凋亡率的假说；(2)利用条件性小鼠ATE1基因敲除方法，鉴定导致ATE1-/-发育表型的细胞系，并验证此表型至少部分是由于神经分泌细胞、内皮细胞系和心肌细胞的变化所致的假说；(3)利用生化分析和质谱学方法，鉴定心血管组织中体内精氨化的蛋白质，并确定ATE1-/-心血管表型的可能分子机制。综上所述，拟议的实验将有助于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。 与公共卫生相关：ATE1-/-心血管表型类似于常见的人类先天性心脏病，包括室间隔和房间隔缺损(VSD和ASD)、持续性动脉干动脉硬化(PTA)和DiGeorge综合征。这项拟议的研究旨在解决这些疾病以前未被探索的机制，并为其治疗的新可能性提供见解。

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.