Post Translational Addition of Amino Acids - Mechanisms Substrate Recognition

蛋白质降解是多种细胞过程的基础，包括基因调控、蛋白水解级联的减弱和错误折叠或受损蛋白质的去除。 我们正在研究被称为N-degron的进化保守降解信号，这是真细菌和真核生物中N末端规则降解的信号。N-末端规则途径是一种普遍存在的过程，其靶向具有特定N-末端氨基酸的蛋白质进行降解。 氨酰-tRNA蛋白转移酶催化来自氨酰-tRNA的不稳定氨基酸翻译后添加到底物蛋白的N-末端，靶向它们进行N-末端规则降解。 为了研究细菌氨酰-tRNA蛋白转移酶（L/F转移酶）的活性，我们最近开发了一种新的基于质谱的程序来定量酶活性。 利用现有的L/F转移酶的X射线结构数据和质谱方法，我们完成了研究，使我们能够提出一种新的催化机制的酶。 我们建议继续我们的工作L/F转移酶，并专注于底物识别。 最近，第一个在体内L/F转移酶底物被确定，并显着地，这种基板缺乏的特征N-末端，将预测从历史上的L/F转移酶的生化数据。 这种差异的生化数据和最近在体内的数据主张系统评价的序列要求底物识别和底物蛋白的额外识别。 本论文将利用现代质谱技术对大肠杆菌的底物序列选择性进行全面的研究。coli L/F转移酶，并使用“点击化学”和蛋白质组学方法来鉴定另外的体内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，以研究E.杆菌

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.