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.

细菌在人类健康中扮演着重要的角色。细菌群落是正常生活的重要组成部分 生理学，由对人类微生物区系的研究揭示的。相比之下，病原菌会导致发病率和 死亡率。无论是影响健康还是影响疾病，细菌与宿主的相互作用都有许多共同之处 功能。为了生存和茁壮成长，细菌必须监测它们的细胞内和细胞外环境，并 对不断变化的条件作出适当的适应性反应。“双组分体系”(TCS)磷转移 涉及传感器组氨酸蛋白激酶和磷酸化激活的反应调节因子的通路 生成的输出响应包括一个通用的监管方案，该方案发生在数十万 监管体系。在核心元件的结构和功能保守的同时，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表明 酶活性大小、大分子相互作用亲和力、信号水平的差异 蛋白质和系统架构，所有这些都可能有助于调整对需求的响应行为 单独的系统。本研究的主要目标是了解交通控制系统的设计原则，以便 利用系统参数的知识可以预测系统行为或至少使系统行为合理化的程度。 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，然而它们在细胞研究中通常被忽视。组氨酸激酶和反应调节因子 众所周知，不同TCS之间的浓度和化学计量有很大的不同，但这些变化的影响 除了健壮性之外，对系统行为的影响在很大程度上还没有得到研究。这个项目将通过探索如何 组氨酸激酶和反应调节剂浓度影响系统设计和行为。调查将会 使用一组19个大肠杆菌TCS进行。方法将利用报告基因分析和 测量反应调节剂的细胞内磷酸化以量化反应输出、质量 未诱导和激活状态下细胞内双组分蛋白质水平的光谱分析 条件，用实验数据进行数学建模以确定动力学参数和预测系统 持续培养中的行为和竞争分析，以评估健康状况。研究将涉及四大领域 问题。TCS调节器的大小是否对TCS中的响应调节器的水平提出了要求？ 组氨酸激酶和反应调节剂的化学计量比如何影响反应输出 激活的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？反应调节剂的非特异性磷酸化是否会使 对组氨酸激酶的磷酸酶活性的要求？这些调查将确定核心设计 TCS的原理以及如何在不同的系统中适应各个参数的变化。 这项关于TCS的研究揭示的原则很可能广泛适用于其他监管系统。