Molecular Mechanisms of Signal Transduction by Two-Component Regulatory Systems

二元调控系统信号转导的分子机制

• 批准号: 7931609
• 负责人: Robert B. Bourret
• 金额: $ 7.1万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931609
• 关键词: Accounting; Active Sites; Affect; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Antibiotic Resistance; Archaea; Bacteria; Bacterial Drug Resistance; Behavior; Binding; Biochemistry; Bioinformatics; Biological; Biological Assay; Biological Process; Biophysics; Cells; Characteristics; Development; Elements; Engineering; Environment; Eukaryota; Excision; Frequencies; Genetic; Genetic Variation; Goals; Growth; Health; Human; Ionic Strengths; Kinetics; Knowledge; Length; Life; Malignant Neoplasms; Measures; Methods; Microbe; Modeling; Molecular; Molecular Biology; OmpR protein; Output; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Plants; Positioning Attribute; Prokaryotic Cells; Property; Protein Dephosphorylation; Protein Family; Protein Kinase; Proteins; Reaction; Regulation; Role; Signal Transduction; Speed; Stimulus; Structure; Substrate Specificity; System; Temperature; Testing; Therapeutic Agents; Time; Translating; Virulence; Water; Work; acetyl phosphate; cell growth; design; gain of function; gain of function mutation; inhibitor/antagonist; innovation; insight; interest; microorganism; mutant; pathogen; reaction rate; response; sensor; small molecule; structural biology

DESCRIPTION (provided by applicant: All living cells use signal transduction to detect properties of interest in their environment, create an internal representation of stimuli, and generate an appropriate response to changing conditions. Errors in signal transduction can have serious consequences, such as cell growth without a growth stimulus (i.e. cancer). In both prokaryotes and eukaryotes, information is often encoded as the presence or absence of a phosphoryl group specifically attached to a protein. Understanding the mechanisms and regulation of phosphoryl group transfer among proteins, and the impact of phosphorylation on protein activity, is therefore of broad interest. Because microorganisms constitute the vast majority of life on Earth in terms of both numbers and genetic diversity, microbes are logical subjects in which to seek fundamental biological principles generally applicable to all forms of life. Two-component regulatory systems are widely used for signal transduction by bacteria, archaea, eukaryotic microorganisms, and plants (but not humans). A sensor kinase protein detects stimuli and converts them to phosphoryl groups, which are transferred to a response regulator protein to control responses such as behavior, development, physiology, or virulence. Our long-term goal is to achieve a comprehensive understanding of two-component signal transduction. In this proposal, we will investigate the mechanisms and kinetics by which response regulators switch between phosphorylated (active) and unphosphorylated (inactive) states. In order to synchronize responses with stimuli, the kinetics of signaling biochemistry must match the timescale of the affiliated biological process. Response regulators can self-catalyze phosphoryl group addition and removal. Auxiliary kinases and phosphatases substantially accelerate response regulator autocatalytic reactions to achieve physiologically appropriate signaling speeds, but do not alter the intrinsic reaction mechanisms. Although all response regulators share a conserved structure and catalytic residues, autodephosphorylation rates vary by >40,000x. Aims 1 and 2 focus on identifying factors that control rates of response regulator self dephosphorylation and phosphorylation, and understanding how these elements exert their influence. Our experimental strategy integrates biochemistry, bioinformatics, genetics, and structural biology to alter nonconserved residues in the active sites of various response regulators and determine the functional and structural consequences of doing so. Aims 3 and 4 investigate several specific auxiliary phosphatases (e.g. CheZ, CheX, PhoR) in detail to determine what common mechanistic, regulatory, or structural features may exist among this poorly characterized group of proteins. Antibiotic resistance is a major and increasing threat to human health. This work may impact design of therapeutic agents to attack two-component systems that control virulence or viability of bacterial and fungal pathogens. In addition, the knowledge gained could be used to predict or manipulate the signaling kinetics of two-component systems, or engineer synthetic regulatory circuits with specific timing characteristics.

描述（由申请人提供：所有活细胞都使用信号转导来检测其环境中感兴趣的特性，创建刺激的内部表示，并对变化的条件产生适当的响应。信号转导的错误可能会带来严重的后果，例如没有生长刺激的细胞生长（即癌症）。因此，了解蛋白质之间的机制和调节，以及磷酸化对蛋白质活性的影响是广泛的，因为微生物构成了地球上绝大多数生命的数量，而遗传多样性则是逻辑上的逻辑受试者，以寻求基本生物学原理。古细菌，真核微生物和植物（但不是人类）。传感器激酶蛋白检测到刺激并将其转换为磷酸基团，这些磷酸基团被转移到响应调节蛋白中以控制诸如行为，发育，生理或毒力等反应。我们的长期目标是对两个组件信号转导的全面理解。在此提案中，我们将研究响应调节剂在磷酸化（活动）和无磷酸化（非活动）状态之间切换的机制和动力学。为了使响应与刺激同步，信号生物化学的动力学必须与附属生物过程的时间尺度相匹配。响应调节器可以自我催化磷酸组的添加和去除。辅助激酶和磷酸酶基本上加速了响应调节剂自催化反应以达到生理上适当的信号速度，但不会改变内在的反应机制。尽管所有响应调节剂具有保守的结构和催化残基，但自动磷酸化率> 40,000倍。目标1和2的重点是识别控制响应调节剂自磷酸化和磷酸化速率的因素，并了解这些元素如何发挥其影响。我们的实验策略整合了生物化学，生物信息学，遗传学和结构生物学，以改变各种响应调节剂的活跃部位中未经保护的残基，并确定这样做的功能和结构后果。目标3和4研究了几种特定的辅助磷酸酶（例如Chez，Chex，Phor），以确定这种表征较差的蛋白质组中可能存在哪些常见机械，调节或结构特征。抗生素耐药性是对人类健康的主要威胁。这项工作可能会影响治疗剂的设计，以攻击控制细菌和真菌病原体的毒力或生存能力的两组分系统。此外，获得的知识可用于预测或操纵两个组件系统的信号动力学，或具有特定时序特征的工程师合成调节回路。