Role of Protein Arginylation in Cardiovascular Development

描述（申请人提供）：项目摘要：本提案将研究一种新发现的翻译后修饰-N-末端蛋白质重基化-在心血管发育中的作用。PI之前的出版物证明，蛋白质腺苷酸化对心血管发育至关重要，敲除蛋白质腺苷酸化酶ATE 1可导致小鼠胚胎死亡和严重心血管表型，与人类最常见的先天性心脏缺陷非常相似。拟议的研究旨在通过以下三个具体目标，全面了解蛋白质乙酰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）为了检验以下假设：腺苷酸化调节细胞迁移和特化，并可能影响心脏发育过程中细胞凋亡的速率，使用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;（2）鉴定负责Ate 1-/-发育表型的细胞谱系，并检验该表型至少部分归因于神经嵴和内皮谱系细胞以及心肌细胞的变化的假设，使用条件性小鼠ATE 1敲除;和（3）使用生化分析和质谱法鉴定心血管组织中体内腺苷酸化的蛋白质，并确定Ate 1-/-心血管表型的可能分子机制。两者合计，拟议的实验将允许确定在心血管发育控制的蛋白酰化的具体途径，并获得深入了解这种控制在基因表达和蛋白质水平的分子机制。 与公共卫生的相关性：Ate 1-/-心血管表型与常见的人类先天性心脏病相似，包括室间隔缺损和房间隔缺损（VSD和ASD）、持续性动脉干炎（PTA）和DiGeorge综合征。拟议的研究旨在解决这些疾病以前未探索的机制，并提供对其治疗新可能性的见解。

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.