Molecular Mechanisms of Signal Transduction by Two-Component Regulatory Systems

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反应。信号转导中的错误会导致严重的后果，如细胞在没有生长刺激的情况下生长（即癌症）。在原核生物和真核生物中，信息通常被编码为特异性附着在蛋白质上的磷酸化基团的存在或缺失。因此，了解磷酸化基团在蛋白质之间转移的机制和调控，以及磷酸化对蛋白质活性的影响，具有广泛的意义。由于微生物在数量和遗传多样性方面构成了地球上绝大多数生命，因此微生物是寻求普遍适用于所有生命形式的基本生物学原理的逻辑学科。双组分调控系统广泛用于细菌、古生菌、真核微生物和植物（但不包括人类）的信号转导。传感器激酶蛋白检测刺激并将其转化为磷酸化基团，磷酸化基团被转移到反应调节蛋白以控制诸如行为、发育、生理或毒性等反应。我们的长期目标是实现对双组分信号转导的全面理解。在本提案中，我们将研究反应调节因子在磷酸化（活性）和非磷酸化（非活性）状态之间切换的机制和动力学。为了使反应与刺激同步，信号生物化学的动力学必须与相关生物过程的时间尺度相匹配。反应调节因子可以自催化磷酰基的加成和去除。辅助激酶和磷酸酶大大加速了反应调节器的自催化反应，以达到生理上适当的信号传导速度，但不改变内在的反应机制。尽管所有的反应调节因子都有一个保守的结构和催化残基，但自去磷酸化率相差10万倍。目的1和2侧重于确定控制应答调节因子自我去磷酸化和磷酸化速率的因素，并了解这些因素如何发挥其影响。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，以改变各种反应调节因子活性位点的非保守残基，并确定这样做的功能和结构后果。目的3和4详细研究了几种特定的辅助磷酸酶（如CheZ、CheX、PhoR），以确定在这组特征不明显的蛋白质中可能存在哪些共同的机制、调节或结构特征。抗生素耐药性是对人类健康日益严重的重大威胁。这项工作可能会影响治疗药物的设计，以攻击控制细菌和真菌病原体的毒力或活力的双组分系统。此外，所获得的知识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。

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.