Molecular Mechanisms of Protein Arginylation

描述(申请人提供)：信号通路中蛋白质的翻译后修饰是体内控制几乎所有生理过程的因子的激活、失活和降解的最关键的控制点之一。这项建议的目标是确定蛋白质精氨酸化的分子机制和体内功能，这是一种非常鲜为人知的翻译后修饰，估计影响25%的细胞蛋白质组。精氨酸化在细胞迁移和发育形态发生中起关键作用，并与细胞代谢、生理和人类疾病的重大事件有关。我们的初步数据有力地表明，精氨酸化通过快速和 它们的活性和蛋白质相互作用的可逆调节，并构成一个真正必要的和动态的生物调节，然而，几乎不知道底物的专一性和该反应的分子机制。这项提案将通过一种综合的方法来解决精氨基转移酶(ATE1)的机制及其底物专一性的调节，这将提供对这一谜团的第一个机械性见解。 翻译后修饰。我们提出的具体目标如下：(1)确定N-末端精氨酸化的位置特异性和性质；(2)测试完整蛋白质内部蛋白质位点的精氨酸化机制；(3)测试细胞成分对不同ATE1亚型精氨酸化的速率和位置特异性的影响，并在体内调节其活性。总之，这些研究将阐明一种具有重大生物学意义的新型翻译后修饰的分子机制。这些研究将最终实现对ATE1活性的差异化调节和生物靶向，对于探索关键的精氨酸化依赖人类疾病的治疗潜力至关重要，包括心脏病、癌症、神经退行性变和衰老。我们的研究结果将为功能精氨酸化分析和在正常生理和疾病的关键过程中对关键蛋白的精氨酸化进行靶向操作开辟新的可能性。

DESCRIPTION (provided by applicant): Posttranslational modification of proteins in signal pathways is one of the most pivotal control points in vivo for the activation, inactivation, and degradation of factors governing nearly all physiological processes. The goal of this proposal is to define the molecular mechanism and in vivo functions of protein arginylation, a very poorly understood post-translational modification that affects an estimated 25% of the cellular proteome. Arginylation plays a key role in cell migration and developmental morphogenesis, and is implicated in major events of cell metabolism, physiology, and human disease. Our preliminary data strongly suggest that arginylation regulates its protein targets through rapid and reversible modulation of their activity and protein interactions and constitutes a truly essential and dynamic biological regulator, however virtually nothing is known about the substrate site specificity and the molecular mechanisms of this reaction. This proposal will address the mechanisms of the arginyl transfer enzyme (ATE1) and the regulation of its substrate specificity through an integrated approach that will provide the first mechanistic insights into this enigmatic posttranslational modification. We propose the following specific aims: (1) To determine site specificity and properties of N- terminal arginylation; (2) To test the mechanisms of arginylation at internal protein sites in intact proteins; and (3) To test the effect of cellular components on he rate and site specificity of arginylation by different ATE1 isoforms and modulate its activity in vivo. Together, the proposed studies will elucidate the molecular mechanism of a novel posttranslational modification with major biological significance. These studies will ultimately enable differential modulation of ATE1 activity and biological targeting, essential for exploring is therapeutic potential in critical arginylation-dependent human conditions, including heart disease, cancer, neurodegeneration, and aging. The results of our studies will open new possibilities of functional arginylation analysis and targeted manipulation of arginylation of key proteins during essential processes in normal physiology and disease.