Molecular mechanisms of bacterial tyrosine phosphorylation

翻译后修饰在细菌生理学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中广泛保守，那就是蛋白质磷酸化。酪氨酸残基的蛋白磷酸化在真核生物中已经得到了很好的证明，并在调节级联反应中发挥了关键作用。原核生物中的酪氨酸磷酸化知之甚少，到目前为止，只有少数几类细菌酪氨酸激酶被鉴定出来。然而，最近的蛋白质组学研究发现，酪氨酸磷酸化是原核生物中一种丰富的翻译后修饰，而在大肠杆菌中的研究强烈表明，基因组中存在未知的副激酶。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。尽管副激酶和蛋白酪氨酸磷酸酶(PTP)保守，但序列比对显示操纵子内存在遗传差异，这表明该系统可能在菌株之间受到顺从调节。初步数据支持这一点，为这一研究计划提供了基础，该研究计划试图以大肠杆菌作为模型系统来确定细菌中酪氨酸磷酸化的分子机制。我假设，通过对细菌酪氨酸磷酸化系统进行生化表征，我将揭示翻译后修饰如何控制对细菌生理学重要的关键细胞通路。首先，我的实验室将使用基于发光和荧光的记者来确定这一新的酪氨酸磷酸化系统的调节机制。其次，我的实验室将通过结合酶学、结构生物学和生物物理表征技术对新鉴定的By-Kinase和PTP进行生物化学表征。第三，我的实验室将研究主要促进剂超家族(MFS)转运蛋白酪氨酸磷酸化依赖调控的结构基础，这些转运蛋白参与各种溶质的外流，包括抗生素、富含Pro的多肽、胆盐和其他抗菌化合物。总之，这些方法对于了解大肠杆菌中酪氨酸磷酸化的分子基础以及揭示副激酶和PTPs与它们的真核对应物之间的功能差异将是非常宝贵的。我预计，这一提议的发现将极大地促进我们对细胞信号及其在细菌适应中所起作用的理解。将利用这些基本知识来确定细菌酪氨酸磷酸化的第一代抑制剂，并发现新的目标，以设计可极大影响人类和动物健康的抗菌素控制战略，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.