Molecular Mechanisms of Signal Transduction by Two-Component Regulatory Systems

描述(申请人提供)：对刺激做出反应的能力通常被认为是生活的一个关键特征。细胞可以检测新的情况，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蛋白质上磷酸基团的特定和瞬时位置来表示信息。信号转导中的错误可能导致疾病(如癌症、糖尿病)，已经开发出药物来阻断异常的信号过程。因此，了解蛋白质磷酸化的机制、调节和影响具有重要的意义，同时对人类健康也具有重要的现实意义。从许多方面来看，微生物是地球上的主要生命形式，包括遗传多样性、原始数量、环境分布和进化经验。因此，在微生物中寻求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双组分调控系统的信号转导，这发生在所有三个系统发育领域的微生物中。在一个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，反应调节器催化传感器激酶(或小分子)的磷酸化，从而启动反应。反应调节因子去磷酸化是由自身催化的或由磷酸酶介导的，结束反应。磷酸基反应的动力学对于使反应与刺激同步是很重要的。基因组测序既是一个挑战(已知蛋白质的数量与可以研究的蛋白质数量之间的差距迅速扩大)，也是一个机遇(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以万计的两组分蛋白质的性质，我们的创新研究策略专注于传感器激酶或反应调节器的保守结构域之间的序列差异(而不是相似性)。我们在前一次拨款期间取得了丰硕的成果，采用了一种综合了生物化学、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。我们确定了对反应调节反应速率有很大影响的因素，但不能解释整个已知范围。我们对Chex机制的阐明，加上我们之前对CHEZ的研究，为反应调节磷酸酶的统一假说奠定了基础。在我们成功的基础上，我们将确定影响反应调节因子的磷酸结合和自磷酸化(AIM 1)、自去磷酸化(AIM 2)和传感器激酶介导的去磷酸化(AIM 3)的因素，并表征潜在的机制。细菌和真菌病原体对抗生素的耐药性是对人类健康的一个主要且日益严重的威胁。我们对小分子与反应调节器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。我们的项目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性的合成调节电路。信号转导的基本原理也可能会出现。

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.