Two-Component System Design Principles

二元系统设计原则

• 批准号: 9922317
• 负责人: ANN M. STOCK
• 金额: $ 43.73万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9922317
• 关键词: Address; Affinity; Antibiotics; Bacteria; Behavior; Biological Assay; Cells; Communicable Diseases; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Elements; Environment; Environmental Monitoring; Escherichia coli; Goals; Health; Human; In Vitro; Individual; Investigation; Kinetics; Knowledge; Mass Spectrum Analysis; Measurement; Monitor; Morbidity - disease rate; Outcome; Output; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Physiology; Play; Proteins; Reaction; Regulon; Reporter Genes; Research; Role; Scheme; Signal Transduction; Signaling Protein; Structure; System; Variant; bacterial community; behavioral response; combat; commensal microbes; design; enzyme activity; extracellular; fitness; host-microbe interactions; human microbiota; mathematical model; mortality; pathogen; pathogenic bacteria; protein-histidine kinase; response; sensor; stoichiometry; system architecture

Bacteria play important roles in human health. Bacterial communities are important components of normal physiology, as revealed by studies of human microbiota. In contrast, pathogenic bacteria cause morbidity and mortality. Whether impacting health or disease, interactions of bacteria with hosts share many common features. To survive and thrive, bacteria must monitor their intracellular and extracellular environments and elicit appropriate adaptive responses to changing conditions. "Two-component system” (TCS) phosphotransfer pathways involving a sensor histidine protein kinase and a phosphorylation-activated response regulator that generates the output response comprise a versatile regulatory scheme that occurs in hundreds of thousands of regulatory systems. While structure and function of core elements are conserved, TCSs display enormous diversity. Data acquired from numerous studies of individual systems as well as global analyses have revealed differences in the magnitudes of enzyme activities, affinities of macromolecular interactions, levels of signaling proteins and system architecture, all of which presumably contribute to tuning response behavior to the needs of individual systems. The overarching goal of this research is to understand design principles of TCSs to the extent that system behavior can be predicted, or at least rationalized, with knowledge of system parameters. Protein concentrations are known to be critical parameters that influence reaction kinetics and outcomes in vitro, yet they are commonly overlooked in cellular studies. Histidine kinase and response regulator concentrations and stoichiometry are known to differ greatly among TCSs, but the effects of these variations on system behavior, other than robustness, are largely unstudied. This project will fill this gap by exploring how histidine kinase and response regulator concentrations impact system design and behavior. Investigations will be performed using a set 19 Escherichia coli TCSs. Approaches will utilize reporter gene assays and measurement of intracellular phosphorylation of response regulators to quantitate response output, mass spectrometry to quantitate levels of two-component proteins in cells under un-induced and activated conditions, mathematical modeling with experimental data to determine kinetics parameters and predict system behavior, and competition assays in continuous cultures to assess fitness. Studies will address four broad questions. Does the size of a TCS regulon place a requirement on the level of response regulator in a TCS? How does the stoichiometry of histidine kinases and response regulators impact response output in an activated TCS? How are system parameters configured to accommodate differences in histidine kinase and response regulator concentrations? Does non-specific phosphorylation of response regulators place a requirement on the phosphatase activity of histidine kinases? These investigations will identify core design principles of TCSs and how variations in individual parameters are accommodated in different systems. Principles uncovered in this study of TCSs are likely to be broadly applicable to other regulatory systems.

细菌在人类健康中起着重要作用。细菌群落是正常的 生理学，如对人类微生物群的研究所揭示的。相比之下，致病菌引起发病， mortality.无论是影响健康还是疾病，细菌与宿主的相互作用都有许多共同点。 功能.为了生存和繁衍，细菌必须监测其细胞内和细胞外环境， 对不断变化的条件做出适当的适应性反应。“双组分系统”（TCS）磷酸转移 涉及传感器组氨酸蛋白激酶和磷酸化激活的反应调节剂的途径， 产生的输出响应包括一个通用的调节方案，发生在成千上万的 监管制度。虽然核心元件的结构和功能是保守的，但TCS显示出巨大的 多样性从对单个系统的大量研究以及全球分析中获得的数据显示， 酶活性的大小、大分子相互作用的亲和力、信号传导水平的差异 蛋白质和系统结构，所有这些都可能有助于调整响应行为以满足需求 个别系统。本研究的总体目标是了解TCS的设计原则， 系统行为可以预测的程度，或至少合理化，与系统参数的知识。 已知蛋白质浓度是影响反应动力学和结果的关键参数， 在体外，它们通常在细胞研究中被忽视。组氨酸激酶和反应调节剂 已知TCS之间的浓度和化学计量存在很大差异，但这些差异的影响 对系统行为的影响，除了鲁棒性之外，在很大程度上还没有研究。本项目将通过探索如何填补这一空白 组氨酸激酶和反应调节剂浓度影响系统设计和行为。调查将 使用19种大肠杆菌TCS进行。方法将利用报告基因测定， 测量细胞内反应调节剂的磷酸化以定量反应输出，质量 在未诱导和活化条件下定量细胞中双组分蛋白质水平的光谱法 条件下，用实验数据建立数学模型，确定动力学参数，预测体系 行为，和竞争测定在连续培养物中评估健身。研究将涉及四个广泛的 问题. TCS调节子的大小是否对TCS中的响应调节子的水平有要求？ 组氨酸激酶和反应调节剂的化学计量如何影响反应输出， 激活TCS？如何配置系统参数以适应组氨酸激酶和 反应调节剂浓度？反应调节因子的非特异性磷酸化是否会使 对组氨酸激酶磷酸酶活性的要求？这些调查将确定核心设计 TCS的基本原理以及不同系统如何适应单个参数的变化。 本研究所揭示的原则可能广泛适用于其他监管制度。