大肠杆菌氨基酰-tRNA合成酶的乙酰化

Aminoacyl-tRNA synthetases (aaRS) catalyze the ligation of amino acids with its cognate tRNA to produce aminoacyl-tRNA as materials of protein biosynthesis. The editing activity of some aaRSs effectively prevents from formation of mis-aminoacyl-tRNA which will carry the wrong amino acid to incorporate to protein. The accuracy of recognition of aaRS to its cognate amino acid and tRNA ensures the fidelity of translation from mRNA to protein. aaRS plays a very important role in the quality control system of protein biosynthesis. Acetylation is an important modification of post-translational on and involved in many critical functions of cells. By Mass Spectrometry (MS) acetylation of leucyl- and arginyl-tRNA synthetase (LeuRS and ArgRS) from Escherichia coli was found. However the effect of acetylation of the LeuRS and ArgRS on their functions still has not been revealed. In this project, from our preliminary MS results we will investigate the effects and mechanisms of acetylated LeuRS and ArgRS from Escherichia coli, including identify the crucial Lys residues of acetylation in the two aaRSs, study on the relationship between acetylation and activity of the two aaRSs, assay the enzymatic kinetic properties of acetylated LeuRS and ArgRS, analyze the affinity of the two modified aaRSs with their cognate tRNAs and investigate the editing function of the acetylated LeuRS. Further we will explore the physiology conditions to produce acetylation of the two aaRSs. This work will help us to understand the dynamical changes of the functions of aaRSs by modification of post translation and expand our knowledge about the role of aaRSs in quality control of protein biosynthesis.

氨基酰-tRNA合成酶（aaRS）催化氨基酰-tRNA的合成，为蛋白质合成提供原料，它还通过编校反应防止错误的氨基酸掺入蛋白质，保证遗传信息准确传递，在蛋白质合成质量控制中起重要作用。乙酰化修饰是重要的蛋白质翻译后修饰，通过修饰可以调节蛋白质的功能。质谱分析发现大肠杆菌aaRS中的亮氨酰-tRNA合成酶(LeuRS)和精氨酰-tRNA合成酶(ArgRS)存在乙酰化修饰，但乙酰化修饰对酶功能的影响从未见报道。本项目将以质谱结果为线索，研究这两种 aaRS的重要位点乙酰化与活力的关系，分别深入研究LeuRS和ArgRS经乙酰化修饰的酶学动力学常数的影响、与tRNA结合能力的变化，和乙酰化修饰后LeuRS的编校功能的改变，探讨这两种酶产生乙酰化修饰的生理条件。该项目将使我们更好地理解aaRS功能的动态变化，拓展我们对aaRS参与蛋白质合成质量控制的认识。

以往的蛋白组学分析表明，很多生物体中的氨基酰-tRNA合成酶（aaRSs）能够通过赖氨酸(Lys)的乙酰化来修饰，在本研究中，过表达和纯化大肠杆菌亮氨酰-tRNA合成酶和精氨酰-tRNA合成酶，通过质谱(MS)发现赖氨酸残基被乙酰化。谷氨酰胺(Gln)扫描突变显示，大肠杆菌亮氨酰-tRNA合成酶上的Lys619, Lys624 Lys809 残基和大肠杆菌精氨酰-tRNA合成酶上的Lys49, Lys126, Lys197, Lys198 and Lys408 可能在酶活性上起重要作用。此外，我们利用一种新的蛋白质表达系统，在特定位点获得了含有乙酰化的赖氨酸(AcK)的酶，并研究了他们的催化活性。这些赖氨酸残基的乙酰化可通过影响氨基酸活化和/或对tRNA的亲和力来影响其氨基酰化活性。体外实验表明，乙酰磷酸（AcP）非酶乙酰化大肠杆菌亮氨酰-tRNA合成酶和精氨酰-tRNA合成酶，并提示西丁类sirtuin去乙酰化酶CobB可以使乙酰化的这两种酶去乙酰化反应。这些发现暗示赖氨酸ε氨基的乙酰化控制氨基酰-tRNA合成酶活性，氨基酰-tRNA合成酶的去乙酰化和乙酰化分别起到活力开关的作用。这是一种最经济、最快速的调节氨基酰-tRNA合成酶活力的方式，通过这种方式来调节蛋白质合成原料氨基酰-tRNA的生成速度，进而调节蛋白质合成的速度。通过本项目支持，在国际刊物上发表4篇研究论文和2篇中文综述。3位研究生获得博士学位。5位外国科学家访问了我们实验室。