Molecular Mechanisms of Signal Transduction by Two-Component Regulatory Systems

描述（由申请人提供）：对刺激作出反应的能力通常被认为是生命的一个关键特征。细胞可以检测新的条件，将信息转换为可用的形式，并执行适当的响应。一种常见的信号转导策略是通过磷酰基在蛋白质上的特异性和瞬时位置来表示信息。信号转导中的错误可导致疾病（例如癌症、糖尿病），并且已经开发了药物来阻断异常信号传导过程。因此，了解蛋白质磷酸化的机制、调节和影响具有根本意义，对人类健康也具有实际意义。 微生物是地球上生命的主要形式，从许多方面来看，包括遗传多样性、原始数量、环境分布和进化经验。因此，在微生物中寻找基本的信号转导原理是合乎逻辑的。我们的长期目标是全面了解信号转导的双组分调控系统，这发生在微生物从所有三个系统发育域。在典型的双组分系统中，传感器激酶检测刺激和自磷酸化。然后，反应调节剂催化来自传感器激酶（或来自小分子）的磷酸化，这开启了反应。反应调节剂去磷酸化，无论是自催化或磷酸酶介导，结束了反应。磷酰基反应的动力学对于使反应与刺激同步是重要的。 基因组测序提出了一个挑战（已知蛋白质数量与可研究蛋白质数量之间的差距迅速扩大）和一个机会（多样化和广泛的序列数据）。为了了解如何仅从序列数据中揭示数万种双组分蛋白质的特性，我们的创新研究策略专注于传感器激酶或反应调节剂保守结构域之间的序列差异（而不是相似性）。在上一个资助期，我们采用了整合生物化学、生物信息学、生物物理学、遗传学、分子生物学和结构生物学的方法。我们确定了极大地影响反应调节剂反应速率的因素，但不能解释整个已知范围。我们阐明的CheX机制，加上我们以前的工作，对CheZ，设置一个统一的假设的反应调节磷酸酶的阶段。在我们成功的基础上，我们将确定影响磷酸供体结合和自磷酸化（目标1），自去磷酸化（目标2）和传感器激酶介导的反应调节剂的去磷酸化（目标3）的因素，并表征潜在的机制。 细菌和真菌病原体的抗生素耐药性是对人类健康的主要且日益严重的威胁。我们对小分子与反应调节剂结合的研究可能会影响治疗剂的设计，以禁用微生物病原体的关键双组分系统。我们项目的结果也可用于预测或操纵双组分系统的信号动力学，或设计具有特定时序特征的合成调节电路。信号转导的基本原理也可能出现。

DESCRIPTION (provided by applicant): The ability to respond to stimuli is often considered to be a key characteristic of life. Cells can detect new conditions, transduce that information into a usable form, and execute an appropriate response. One common signal transduction strategy is to represent information by the specific and transient placement of phosphoryl groups on proteins. Errors in signal transduction can lead to diseases (e.g. cancer, diabetes), and drugs have been developed to block aberrant signaling processes. Understanding the mechanisms, regulation, and impact of protein phosphorylation is thus of fundamental interest, as well as of practical significance to human health. Microorganisms are the dominant form of life on Earth by many measures, including genetic diversity, raw numbers, environmental distribution, and evolutionary experience. Thus, it is logical to seek basic signal transduction principles in microbes. Our long-term goal is comprehensive understanding of signal transduction by two-component regulatory systems, which occur in microorganisms from all three phylogenetic domains. In a typical two-component system, a sensor kinase detects stimuli and autophosphorylates. A response regulator then catalyzes phosphorylation from the sensor kinase (or from small molecules), which turns on the response. Response regulator dephosphorylation, either self-catalyzed or mediated by a phosphatase, ends the response. The kinetics of phosphoryl group reactions are important to synchronize responses with stimuli. Genome sequencing presents a challenge (a rapidly widening gap between the number of known proteins and what can be studied) and an opportunity (diverse and extensive sequence data). To learn how to reveal properties of tens of thousands of two-component proteins from sequence data alone, our innovative research strategy focuses on sequence differences (rather than similarities) between the conserved domains of sensor kinases or response regulators. We were productive during the previous grant period with an approach that integrated biochemistry, bioinformatics, biophysics, genetics, molecular biology, and structural biology. We identified factors that greatly affect response regulator reaction rates, but do not account for the entire known range. Our elucidation of the CheX mechanism, together with our previous work on CheZ, set the stage for a unified hypothesis of response regulator phosphatases. Building on our success, we will identify factors that affect phosphodonor binding and autophosphorylation (Aim 1), autodephosphorylation (Aim 2), and sensor kinase-mediated dephosphorylation (Aim 3) of response regulators and characterize underlying mechanisms. Antibiotic resistance of bacterial and fungal pathogens is a major and increasing threat to human health. Our study of the binding of small molecules to response regulators may influence design of therapeutic agents to disable critical two-component systems of microbial pathogens. The results of our project could also be used to predict or manipulate the signaling kinetics of two-component systems, or engineer synthetic regulatory circuits with specific timing characteristics. Fundamental principles of signal transduction may also emerge.