Arginyl-tRNA beyond translation: mechanism and regulation of protein arginylation

项目摘要 转移RNA(TRNA)在核糖体介导的蛋白质合成中发挥着重要作用。但是，在一个 精氨酰-tRNA的作用鲜为人知，是催化一种独特的、知之甚少的蛋白质所必需的。 翻译修饰，即精氨酸化，调节蛋白质周转。在这个精氨酸化反应中， ATE1(精氨酰转移酶1)通过一种机制促进精氨酸转移到蛋白质靶点 依赖于精氨酰-tRNA(Arg)作为供体辅因子，并对其具有选择性。ATE1介导的蛋白质 精氨酸化存在于数百种蛋白质上，被认为是真核生物的全球调节因子。 细胞过程，包括胚胎发生、应激反应和衰老。ATE1被发现放松管制 帕金森氏症和转移性前列腺癌、肝癌和皮肤癌的患者。尽管如此，如何 ATE1(和其他氨酰-tRNA转移酶)劫持高效核糖体蛋白中的tRNA 精氨酸化反应的合成途径和催化仍是一个谜。这项建议旨在 阐明ATE1介导的蛋白质精氨酸化在体外和体内的催化机制和调节 细胞。我们将集中在询问ATE1的活性和精氨酸化的结果在三 比例。首先，我们将确定ATE1选择精氨酰-tRNA(Arg)和 通过一种综合方法识别蛋白质靶标中的特定位点， 生物化学和生物物理方法。一旦确定，这项研究将使人们更好地理解 在不断增长的氨酰基-tRNA转移酶类中。第二，我们将在数量上 确定精氨酸化对活细胞中目标蛋白周转的影响。蛋白质降解 通常取决于多泛素化，精氨酸化后的下游或同时发生的事件，以及 通过蛋白酶体或自噬-溶酶体途径。通过检查特定的模型衬底 对于正常或应激条件下的蛋白酶体或自噬，我们将确定串扰。 精氨酸化和泛素化之间的关系；描述每条降解途径的贡献；揭示 细胞内的动力学。最后，我们将调查核糖体的核心成分是否以及如何 翻译机制和营养物质会影响蛋白质的精氨酸化。从机制上讲，这些研究将扩大 我们对氨基酸和tRNAs调控作用的了解，丰富了我们研究的工具箱 通过tRNA依赖的氨酰化来调节大分子，并重塑我们如何看待 带电tRNA的功能超越了蛋白质的合成。总而言之，这项研究提供了基本的 精氨酸化知识，为发现新的治疗策略奠定基础 调节帕金森氏病和转移性癌症的ATE1活性和蛋白精氨酸化，以及 使我们能够为未来的研究建立综合平台。

Project Summary Transfer RNA (tRNA) is best known to function in ribosome-mediated protein synthesis. However, in a less known role, arginyl-tRNA is essential for catalyzing a unique and poorly understood protein post- translational modification, namely arginylation, that regulates protein turnover. In this arginylation reaction, ATE1 (Arginyltransferase 1) facilitates arginine transfer to protein targets using a mechanism that depends on, and is selective for, arginyl-tRNA(Arg) as the donor cofactor. ATE1-mediated protein arginylation was identified on hundreds of proteins and is recognized as a global regulator of eukaryotic cellular processes, including embryogenesis, stress responses, and aging. Deregulation of ATE1 is found in patients with Parkinson’s disease and metastatic prostate, liver, and skin cancers. Nonetheless, how ATE1 (and other aminoacyl-tRNA transferases) hijacks tRNA from the highly efficient ribosomal protein synthesis pathways and catalyzes the arginylation reaction remains a mystery. This proposal aims to elucidate the catalytic mechanism and regulation of ATE1-mediated protein arginylation in vitro and in cells. We will focus on interrogating the activity of ATE1 and the consequences of arginylation at three scales. Firstly, we will determine the molecular mechanisms ATE1 selects for arginyl-tRNA(Arg) and recognizes specific sites in protein targets through an integrative approach combining structural, biochemical, and biophysical methods. Once determined, this research will allow a better understanding of the growing classes of aminoacyl-tRNA transferases in general. Secondly, we will quantitatively determine the consequences of arginylation on target protein turnover in living cells. Protein degradation usually depends on poly-ubiquitination, a downstream or concurrent event following arginylation, and through either proteasomal or autophagy-lysosomal pathways. By examining specific model substrates for proteasome or autophagosome under normal or stressed conditions, we will determine the crosstalk between arginylation and ubiquitination; delineate the contribution of each degradation pathway; reveal the kinetics in cells. Lastly, we will investigate whether and how core components of the ribosomal translation machinery and nutrients affect protein arginylation. Mechanistically, these studies will expand our knowledge of the regulatory roles of amino acids and tRNAs, enrich our toolbox to study macromolecule regulation by tRNA-dependent aminoacylation, and reshape how we consider the functions of the charged tRNAs beyond protein synthesis. Together, this research provides fundamental knowledge about arginylation, lays the groundwork for discovering novel therapeutic strategies by modulating ATE1 activity and protein arginylation in Parkinson’s disease and metastatic cancers, and enables us to build integrative platforms for future research.