Two-Component System Design Principles

二元系统设计原则

• 批准号: 10398849
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10398849
• 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重要作用。细菌群落是正常的重要组成部分 人类微生物群的研究表明，生理学。相反，致病细菌会导致发病率和 死亡。无论是影响健康还是疾病，细菌与宿主的相互作用共享许多常见 特征。为了生存和繁殖，细菌必须监测其细胞内和细胞外环境， 引起对变化条件的适当自适应反应。 “两个组件系统”（TCS）磷酸转移 涉及传感器组氨酸蛋白激酶和磷酸化激活反应调节剂的途径，该途径 产生输出响应包括一种多功能调节方案，该方案发生在数十万个 监管系统。虽然配置了核心元素的结构和功能，但TCSS显示了巨大的 多样性。从单个系统以及全球分析的众多研究中获得的数据揭示了 酶活性量的差异，大分子相互作用的亲和力，信号的水平 蛋白质和系统体系结构，所有这些都可能有助于调整需求的响应行为 单个系统。这项研究的总体目标是了解TCSS的设计原理 具有系统参数的知识可以预测系统行为的程度。 已知蛋白质浓度是影响反应动力学和结果的关键参数 组氨酸激酶和反应调节剂，但在细胞研究中通常会忽略它们。 众所周知，浓度和化学计量在TCS中有所不同，但是这些变化的影响 除了鲁棒性以外的系统行为，在很大程度上没有研究。这个项目将通过探索如何填补这一空白 组氨酸激酶和响应调节剂浓度会影响系统的设计和行为。调查会 使用19个大肠杆菌TCSS进行。方法将利用记者基因分析和 测量响应调节剂细胞内磷酸化以定量响应输出，质量 在未诱导和激活的细胞中定量细胞中两个分量蛋白水平的光谱法 条件，使用实验数据的数学建模，以确定动力学参数并预测系统 行为和连续文化中的竞争分析以评估健身。研究将解决四个广泛 问题。 TCS的大小是否对TCS中的响应调节器级别提出了要求？ 组氨酸激酶和反应调节剂的化学计量如何影响反应输出 激活的TCS？如何配置系统参数以适应组氨酸激酶的差异和 响应调节剂浓度？反应调节器的非特异性磷酸化是否放置 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系统中适应各个参数的变化。 TCSS研究中发现的原则可能广泛适用于其他监管系统。