Molecular Mechanisms of Signal Transduction by Two-Component Regulatory Systems

项目总结 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。细胞可以检测到新的 条件，将该信息转换为可用的形式，并执行适当的响应。一个共同之处 信号转导策略是通过特定和瞬时的磷酸化来表达信息 蛋白质基团。信号转导错误可能导致疾病(如癌症、糖尿病)，而药物 被开发用来阻止异常的信号传递过程。了解机制、监管和影响 因此，蛋白质磷酸化的研究不仅对人类健康具有实际意义，而且具有根本的意义。 从许多方面来看，微生物是地球上主要的生命形式，包括遗传多样性、原始生物 数量、环境分布和进化经验。因此，寻求基本信号是合乎逻辑的 微生物的转导原理。我们的长期目标是全面了解信号转导 由来自所有三个系统发育域的微生物中出现的两组分调控系统，如 以及植物。在一个基本的双组分系统中，传感器激酶(SK)检测刺激和自磷酸化。 使用三磷酸腺苷。然后，响应调节器(RR)催化来自SK(或来自小分子)的磷转移， 这就开启了反应。RR去磷酸化，要么是自己催化的，要么是被另一种蛋白质刺激的， 结束响应。包含组氨酸磷酸转移酶(HPT)蛋白会导致更多 通过在基本的SK到RR方案上增加一个HPT和第二个RR，实现了复杂的多步磷继电器。这个 磷酰基反应的动力学和方向性对于使反应与刺激同步很重要。 基因组测序是一项挑战(已知蛋白质数量之间的差距迅速扩大 以及什么可以通过实验进行研究)和机会(多样化和广泛的序列数据)。去学习 如何仅从序列数据中揭示数十万种双组分蛋白质的性质，我们的 创新的研究策略专注于不同物种之间的氨基酸序列差异(而不是相似性) SKS、HPTS或RRS的保守结构域。我们久负盛名且富有成效的实验方法 集生物化学、生物信息学、生物物理学、分子生物学和结构生物学于一体。在这个项目中， 我们将确定影响自身催化的RR磷酸化和去磷酸化(AIM)动力学的因素 1)，SK刺激的RRs去磷酸化(AIM 2)，以及HPTS和RRs之间的磷酸转移反应 (目标3)。我们还将描述这些反应背后的分子机制。 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。 RRs是双组分体系中大多数磷转移反应的中心。我们对小分子的结合的研究 RRS的分子可能会影响治疗剂的设计，使关键的双组分系统失效 微生物病原体。我们项目的结果也可以用来预测或操纵信号 二组分体系的动力学。信号转导的基本原理也可能会出现。

PROJECT SUMMARY 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, as well as plants. In a basic two-component system, a sensor kinase (SK) detects stimuli and autophosphorylates using ATP. A response regulator (RR) then catalyzes phosphotransfer from the SK (or from small molecules), which turns on the response. RR dephosphorylation, either self-catalyzed or stimulated by another protein, ends the response. Inclusion of histidine-containing phosphotransferase (Hpt) proteins results in more complex multi-step phosphorelays by adding an Hpt and a second RR onto the basic SK to RR scheme. The kinetics and directionality 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 experimentally) and an opportunity (diverse and extensive sequence data). To learn how to reveal properties of hundreds of thousands of two-component proteins from sequence data alone, our innovative research strategy focuses on amino acid sequence differences (rather than similarities) between the conserved domains of SKs, Hpts, or RRs. Our well-established and productive experimental approach integrates biochemistry, bioinformatics, biophysics, molecular biology, and structural biology. In this project, we will identify factors that affect the kinetics of self-catalyzed RR phosphorylation and dephosphorylation (AIM 1), SK-stimulated dephosphorylation of RRs (AIM 2), and phosphotransfer reactions between Hpts and RRs (AIM 3). We will also characterize the molecular mechanisms underlying each of these reactions. Antibiotic resistance of bacterial and fungal pathogens is a major and increasing threat to human health. RRs are central to most phosphotransfer reactions of two-component systems. Our study of binding of small molecules to RRs 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. Fundamental principles of signal transduction may emerge as well.