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广泛保守的整个王国的生活是蛋白质磷酸化。酪氨酸残基的蛋白质磷酸化在真核生物中已经有很好的记录，并且在调节级联中起着关键作用。原核生物中的酪氨酸磷酸化知之甚少，迄今为止仅鉴定了几类细菌酪氨酸（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E。coli中的结果强烈表明基因组中存在未知的BY激酶。在我的博士后研究中，我在大肠杆菌中发现了一个新的细菌酪氨酸磷酸化系统。在实验室、实验室和致病菌株中保守的大肠杆菌。尽管保守的BY-激酶和蛋白酪氨酸磷酸酶（PTP），序列比对显示操纵子内的遗传变异，这表明该系统可以在菌株之间去乙酰化调节。初步的数据支持这一点，为这项研究计划提供了基础，该研究计划旨在利用大肠杆菌确定细菌中酪氨酸磷酸化的分子机制。大肠杆菌作为模型系统。我假设，通过表征细菌酪氨酸磷酸化系统的生物化学，我将揭示如何翻译后修饰控制细菌生理学重要的关键细胞通路。 首先，我的实验室将确定这种新的酪氨酸磷酸化系统的调控机制，使用发光和荧光为基础的报告。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进行生物化学表征。第三，我的实验室将研究酪氨酸磷酸化依赖性调节主要易化超家族（MFS）转运蛋白的结构基础，这些转运蛋白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将是非常宝贵的。大肠杆菌和揭示BY-激酶和PTP的功能差异相比，他们的真核生物。我预计，这项提议的发现将大大促进我们对细胞信号及其在细菌适应中所起作用的理解。这些基本知识将被用来识别细菌酪氨酸磷酸化的第一代抑制剂，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.