Molecular mechanisms of bacterial tyrosine phosphorylation

翻译后修饰在细菌生理中起着关键的调节作用。一个众所周知的翻译后修饰在整个生命王国广泛保守是蛋白质磷酸化。酪氨酸残基的蛋白磷酸化已经在真核生物中得到了很好的记录，并且在调节级联反应中起着关键作用。人们对原核生物中酪氨酸的磷酸化作用了解甚少，迄今为止仅鉴定出几种细菌酪氨酸（BY）激酶。然而，最近的蛋白质组学研究已经确定酪氨酸磷酸化是原核生物中丰富的翻译后修饰，并且在大肠杆菌中的研究强烈表明基因组中存在未知的by激酶。在我的博士后研究期间，我在大肠杆菌中发现了一种新的细菌酪氨酸磷酸化系统，该系统在实验室，共生和致病菌株中都是保守的。尽管by激酶和蛋白酪氨酸磷酸酶（PTP）保存，序列比对显示操纵子内的遗传变异，表明该系统可能在菌株之间受到不同的调节。初步数据支持这一点，为本研究计划提供了基础，该研究计划旨在确定以大肠杆菌为模型系统的细菌中酪氨酸磷酸化的分子机制。我假设，通过生物化学表征细菌酪氨酸磷酸化系统，我将揭示翻译后修饰如何控制细菌生理学重要的关键细胞途径。首先，我的实验室将使用基于发光和荧光的报告器确定这种新的酪氨酸磷酸化系统的调节机制。其次，我的实验室将通过酶学、结构生物学和生物物理表征技术的结合，对新发现的by激酶和PTP进行生化表征。第三，我的实验室将研究酪氨酸磷酸化依赖性调节的主要促进剂超家族（MFS）转运体的结构基础，这些转运体参与各种溶质的外排，包括抗生素、富含脯氨酸的肽、胆汁盐和其他抗菌化合物。总之，这些方法对于理解大肠杆菌中酪氨酸磷酸化的分子基础以及揭示by激酶和PTPs与真核生物的功能差异将是非常宝贵的。我预计，这一建议的发现将大大促进我们对细胞信号传导及其在细菌适应中所起作用的理解。这一基础知识将被用来识别第一代细菌酪氨酸磷酸化抑制剂，并发现设计抗菌控制策略的新目标，这些策略可以极大地影响人类和动物的健康，使加拿大人和我们的农业部门受益。

Post-translational modifications play a key regulatory role in bacterial physiology. A well known post-translational modification broadly conserved across the kingdom of life is protein phosphorylation. Protein phosphorylation of tyrosine residues has been well documented in eukaryotes, and plays a key role in regulatory cascades. Tyrosine phosphorylation in prokaryotes is poorly understood, and only a few classes of Bacterial tYrosine (BY)-kinases have been identified to date. However, recent proteomic studies have identified tyrosine phosphorylation as an abundant post-translational modification in prokaryotes, and studies in E. coli strongly suggest that unknown BY-kinases exist in the genome. During my postdoctoral studies I identified a new bacterial tyrosine phosphorylation system in E. coli that is conserved throughout laboratory, commensal, and pathogenic strains. Despite conservation of the BY-kinase and Protein Tyrosine Phosphatase (PTP), sequence alignments show genetic variation within the operon suggesting that  this system may be deferentially regulated between strains. Preliminary data supports this, providing the basis for this research program that seeks to determine the molecular mechanisms of tyrosine phosphorylation in bacteria using E. coli as a model system. I hypothesize that by characterizing bacterial tyrosine phosphorylation systems biochemically, I will uncover how post-translational modifications control key cellular pathways important for bacterial physiology. First, my lab will determine the mechanism of regulation of this new tyrosine phosphorylation system using luminescence- and fluorescence-based reporters. Secondly, my lab will biochemically characterize the newly identified BY-kinase and PTP through a combination of enzymology, structural biology, and biophysical characterization techniques. Third, my lab will investigate the structural basis for tyrosine phosphorylation-dependent regulation of major facilitator superfamily (MFS) transporters that are involved in the efflux of a variety of solutes including antibiotics, proline-rich peptides, bile salts, and other antimicrobial compounds. Together, these approaches will be invaluable for understanding the molecular basis for tyrosine phosphorylation in E. coli and uncover the functional differences between BY-kinases and PTPs compared to their eukaryotic counterparts. I anticipate that discoveries made by this proposal will significantly advance our understanding of cellular signaling and the role it plays in bacterial adaptation. This fundamental knowledge will be harnessed to identify first generation inhibitors of bacterial tyrosine phosphorylation and uncover novel targets for the design of antimicrobial control strategies that can greatly impact human and animal health, benefiting Canadians and our agriculture sector.