Post Translational Addition of Amino Acids - Mechanisms Substrate Recognition

蛋白质降解是各种细胞过程的基础，这些过程包括基因调节、蛋白水解级联的减弱和错误折叠或受损蛋白质的去除。我们正在研究进化保守的降解信号N-degron，它是真细菌和真核生物中N-末端规则降解的信号。N-末端规则途径是一个普遍存在的过程，它针对具有特定N-末端氨基酸的蛋白质进行降解。氨酰-tRNA蛋白转移酶催化底物蛋白质N-末端的不稳定氨基酸的翻译后加成，靶向底物蛋白质的N-末端降解。 为了研究细菌氨酰-tRNA蛋白转移酶L/F转移酶的活性，我们最近建立了一种新的基于质谱学的酶活性定量方法。利用已有的L/F转移酶的X射线结构数据和OUT-MS方法，我们已经完成了研究，从而提出了该酶的一种新的催化机理。 我们建议继续我们对L/F转移酶的研究，并专注于底物识别。最近，第一个体内L/F转移酶底物被鉴定出来，该底物明显缺乏从L/F转移酶的历史生化数据中预测的特征N末端。生化数据和最近的活体数据的这种差异表明，需要对底物识别和底物蛋白质的额外鉴定的序列要求进行系统的评估。 利用现代质谱学技术，我们将全面研究大肠杆菌L/F转移酶的底物序列选择性，并利用点击化学和蛋白质组学方法在体内鉴定额外的L/F转移酶底物。我们还将扩大我们对L/F转移酶的重点研究，以检查整个大肠杆菌中蛋白质周转的各个方面。

Protein degradation is fundamental to a variety of cellular processes which include gene regulation, attenuating of proteolytic cascades and the removal of mis-folded or damaged proteins. We are investigating the evolutionary conserved degradation signal known as the N-degron, which is the signal for N-End Rule degradation in both eubacteria and eukaryotes. The N-end rule pathway is a ubiquitous process that targets proteins for degradation that possess a particular N-terminal amino acid. Aminoacyl-tRNA protein transferases catalyze the post-translational addition of destabilizing amino acids from aminoacyl-tRNAs to the N-termini of substrate proteins, targeting them for N-End rule degradation. To investigate the activity of the bacterial aminoacyl-tRNA protein transferase, L/F transferase, we have recently developed a novel mass spectrometry based procedure to quantify enzymatic activity. With the available X-ray structural data available for L/F transferase and out mass spectrometry method we have completed investigations that have enabled us to propose a new catalytic mechanism for the enzyme. We propose to continue our work on L/F transferase and focus on substrate recognition. Recently the first in vivo L/F transferase substrate was identified and remarkably this substrate lacks the characteristic N-termini that would predicted from historic biochemical data on L/F transferase. This discrepancy of biochemical data and recent in vivo data argues for a systematic evaluation of sequence requirements for substrate recognition and the additional identification of substrate proteins. With modern mass spectrometry techniques we will comprehensively investigate the substrate sequence selectivity of E. coli L/F transferase and use 'click chemistry' and proteomic approaches to identify additional in vivo L/F transferase substrates. We will also broaden our focused investigations on L/F transferase to examine aspects of protein turnover as a whole in E. coli.