Interactions between Bacterial Tyrosine Kinases and Phosphatases

细菌酪氨酸激酶和磷酸酶之间的相互作用

• 批准号: 8359274
• 负责人: RANAJEET GHOSE
• 金额: $ 24.54万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-07 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8359274
• 关键词: Affect; Anti-Bacterial Agents; Antibiotic Resistance; Bacteria; Bacterial Physiology; Binding; Biochemical; Biological Assay; C-terminal; Catalytic Domain; Cell physiology; Cells; Characteristics; Desiccation; Development; Enzymes; Equilibrium; Escherichia coli; Escherichia coli K12; Eukaryota; Face; Family; Goals; Gram-Positive Bacteria; Human; In Vitro; Individual; Knowledge; Laboratories; Lead; Length; Measurement; Measures; Mediating; Methods; Microbial Biofilms; Modeling; Molecular Weight; Mutation; NMR Spectroscopy; Nature; Organism; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Physiology; Play; Polysaccharides; Process; Production; Protein Dephosphorylation; Protein Kinase; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Regulation; Relaxation; Research; Research Project Grants; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Solutions; Spin Labels; Staphylococcus aureus; Streptococcus pneumoniae; Stress; Structure; Surface; System; Tail; Testing; Tyrosine; Tyrosine Phosphorylation; Virulence; Virulence Factors; Walkers; base; capsule; colanic acid; design; enteropathogenic Escherichia coli; genetic manipulation; in vivo; insight; mutant; novel; pathogen; protein folding; response; therapeutic target

DESCRIPTION (provided by applicant): Tyrosine phosphorylation, long studied in eukaryotes, is beginning to be recognized as playing a key regulatory role in bacterial physiology. This process is catalyzed by a novel class of protein tyrosine kinases, the BY- kinases, which are conserved in both Gram-negative and Gram-positive bacteria, but are distinct from their eukaryotic counterparts, with unique structural and regulatory features. BY-kinase mediated signal transduction plays a central role in the synthesis of polysaccharides responsible for capsule and biofilm formation, the former acting as antiphagocytic virulence factors for many pathogenic species, and the latter protecting the cell from a variety of environmental stresses. The regulatory module for BY-kinases is a cluster of tyrosine residues (YC) in the C-terminal tail of its cytosolic catalytic domain. The YC is autophosphorylated in an intermolecular fashion and dephosphorylated by low molecular weight protein tyrosine phosphatases (LMW-PTP). Unlike eukaryotic protein kinases which act as active/inactive or "on-off" switches dependent on the quantitative phosphorylation states of one or more regulatory residues, in BY-kinases both phosphorylated and dephosphorylated species are required for optimal polysaccharide synthesis. Therefore it appears that the dynamic equilibrium between phosphorylated and dephosphorylated forms, resulting from a balance between the kinase and phosphatase activities, is key to proper cellular function. Thus, determining the nature of the interactions between BY-kinases and LMW-PTPs is of fundamental importance in fully understanding the physiochemical aspects of tyrosine phosphorylation mediated signaling in bacteria. Although the structures of several BY-kinase catalytic domains and bacterial LMW-PTPs are available, these do not provide any insight into the nature of their mutual interactions or the physiological consequences of disrupting them. Using the catalytic domain of Wzc, an E. coli (K12) BY-kinase and Wzb, the corresponding LMW-PTP, as a model kinase-phosphatase system, and utilizing state-of-the-art solution NMR methods combined with site-directed mutagenesis, we will ascertain the key residues that define the kinase- phosphatase interface. The relevance of these NMR-determined interactions in the context of the physiology of the intact host organism will be ascertained by assaying cells expressing mutant proteins for polysaccharide synthesis and survival under desiccation. We expect that our present studies, limited to the E. coli Wzc/Wzb system, will facilitate the development of a more elaborate research project involving other bacterial BY-kinase/LMW-PTP pairs including those from pathogenic species, and the construction of a comprehensive mechanistic model of BY-kinase mediated tyrosine phosphorylation in bacteria. PUBLIC HEALTH RELEVANCE: The regulation of BY-kinases by their corresponding phosphatases is being recognized as a key determinant of the virulence of many pathogenic bacterial species. The results of our research that aims to elucidate this regulatory mechanism should facilitate the design of antibacterial therapeutics targeting this pathway.

描述(申请人提供)：酪氨酸磷酸化，在真核生物中长期研究，开始被认为在细菌生理学中起着关键的调节作用。这一过程是由一类新的蛋白酪氨酸激酶催化的，这种蛋白酪氨酸激酶在革兰氏阴性和革兰氏阳性细菌中都是保守的，但不同于真核生物，具有独特的结构和调节功能。By-Kinase介导的信号转导在多糖的合成中起着核心作用，这些多糖负责形成被膜和生物膜，前者作为许多病原菌的抗吞噬毒力因子，后者保护细胞免受各种环境胁迫的伤害。副激酶的调节模块是其胞质催化结构域C末端的一簇酪氨酸残基(YC)。YC以分子间方式自动磷酸化，并被低分子蛋白酪氨酸磷酸酶(LMW-PTP)去磷酸化。与依赖于一个或多个调节残基的定量磷酸化状态而充当活性/非活性或“开-关”开关的真核蛋白激酶不同，在副酶中，磷酸化和去磷酸化物种都是最佳多糖合成所必需的。因此，磷酸化和去磷酸化形式之间的动态平衡似乎是细胞正常功能的关键，这种动态平衡是由激酶和磷酸酶活性之间的平衡产生的。因此，确定副激酶和LMW-PTPs之间相互作用的性质对于全面理解细菌中酪氨酸磷酸化介导的信号转导的物理化学方面是至关重要的。虽然已经有了几个辅酶催化结构域和细菌LMW-PTP的结构，但这些结构并不能提供任何关于它们相互作用的性质或破坏它们的生理后果的见解。我们将利用WZC的催化结构域和相应的LMW-PTP，结合定点突变和最新的溶液核磁共振方法，确定决定激酶-磷酸酶界面的关键残基。这些核磁共振确定的相互作用在完整宿主生物体的生理背景下的相关性将通过检测表达突变蛋白的细胞来确定，这些突变蛋白用于多糖的合成和在干燥条件下的生存。我们希望，我们目前的研究局限于大肠杆菌WZC/WZB系统，将有助于开展包括病原菌在内的其他细菌BYK/LMW-PTP对的更复杂的研究项目，并构建BYK介导的细菌酪氨酸磷酸化的全面机制模型。 与公共卫生相关：许多致病细菌物种的毒力的关键决定因素是由其相应的磷酸酶调节的副激酶。我们的研究结果旨在阐明这一调控机制，应该有助于针对这一途径的抗菌治疗药物的设计。