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属性，创建刺激的内部表示，并对不断变化的条件产生适当的反应。信号转导中的错误可能会产生严重的后果，例如在没有生长刺激的情况下细胞生长(即癌症)。在原核生物和真核生物中，信息通常被编码为特定结合在蛋白质上的磷酸基的存在或不存在。因此，了解蛋白质间磷酸基转移的机制和调节，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生命的绝大多数，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合乎逻辑的主题。两组分调控系统被广泛用于细菌、古菌、真核微生物和植物(但不是人类)的信号转导。传感器激酶蛋白检测刺激并将其转化为磷酸基，这些基团被转移到反应调节蛋白以控制反应，如行为、发育、生理或毒力。我们的长期目标是全面了解双组分信号转导。在这项提案中，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。响应调节剂可以自我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，以达到生理上合适的信号速度，但不改变固有的反应机制。尽管所有的反应调节子都有一个保守的结构和催化残基，但自动去磷酸化速率因&gt；40,000倍而异。目标1和2的重点是确定控制反应调节器自身去磷酸化和磷酸化速率的因素，并了解这些因素是如何发挥影响的。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器活性部位的非保守残基，并确定这样做的功能和结构后果。目标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佳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的一个主要且日益严重的威胁。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生存能力的双组分系统。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性的合成调节电路。

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.