Molecular principles of stringent response activation in bacteria

细菌严格反应激活的分子原理

• 批准号: 10453921
• 负责人: Andrei Korostelev
• 金额: $ 53.42万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-05 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10453921
• 关键词: Acyl Carrier Protein; Address; Adult; Affect; Amino Acids; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Architecture; Bacteria; Bacterial Infections; Binding; Biochemical; Biochemistry; Biological Assay; C-terminal; Catalytic Domain; Cause of Death; Childhood; Codon Nucleotides; Complex; Cryoelectron Microscopy; Crystallography; Data; Development; Drug resistance; Electron Microscopy; Enzymes; GTP Pyrophosphokinase; Genetic Transcription; Gram-Negative Bacteria; Guanosine Triphosphate Phosphohydrolases; Health; Homologous Gene; Hydrolase; Hydrolysis; Immune response; Infection; Kinetics; Lead; Length; Ligase; Measures; Mediating; Messenger RNA; Methodology; Methods; Molecular; Molecular Conformation; N-terminal; Nucleotides; Organism; Pathogenesis; Pathogenicity; Pathway interactions; Play; Proteins; REL Protein; RNA; Regulation; Ribosomes; Role; Salmonella; Second Messenger Systems; Site; Spottings; Stress; Structure; Testing; Transfer RNA; Translating; Work; biological adaptation to stress; chronic infection; computerized data processing; cryogenics; data acquisition; deprivation; improved; infectious disease treatment; inhibitor/antagonist; innovation; monomer; mortality; mutant; novel therapeutic intervention; novel therapeutics; nutrient deprivation; pathogen; pathogenic bacteria; prevent; protein structure; response; stressor; therapeutic target; tool

PROJECT SUMMARY/ABSTRACT As the leading cause of childhood mortality and a major cause of adult mortality, bacterial infections remain the main threat to health worldwide. The emergence of antibiotic resistance is outpacing the development of new antibiotics and posing a serious challenge to infectious disease treatment. Understanding how bacteria adapt to stress during infection will spur the search for novel therapeutic approaches. To become therapeutic targets, these mechanisms have to play key functional roles in pathogens but not in mammalian organisms. Bacterial “stringent response”—the master regulator of bacterial stress adaptation—drives bacterial pathogenesis and antibiotic resistance. Stringent response depends on RelA-like proteins that make and regulate the second messenger molecules pppGpp and ppGpp, which reformat cellular transcription to adapt to stresses. RelA-like proteins, such as RelA and SpoT exist in most pathogenic bacteria. Despite many cellular and biochemical studies, the mechanisms of activation of RelA-like proteins and stringent response remain poorly understood. How RelA-like proteins sense stress and catalyze pppGpp synthesis and hydrolysis is unclear. RelA is generally thought to be activated by deacyl-tRNAs, which accumulate in response to amino acid deprivation and bind specific mRNA codons. Paradoxically, many other types of stress activate stringent response, and our preliminary biochemical and structural studies suggest that RelA and SpoT can be activated by ribosomes in the absence of deacyl-tRNA. The proposed studies will test the paradigm-shifting hypothesis that RelA-like proteins sense many stresses via ribosome stalling, and that ribosomes and other activators induce distinct conformational changes in RelA and SpoT to regulate their activities. This project will take advantage of recent methodological innovations and advances in biochemistry and cryogenic electron microscopy (cryo-EM) to dissect the molecular mechanisms of two RelA-like proteins. Aim 1 will dissect the biochemical basis of the stringent response activation by RelA by identifying the functions of regulatory domains and by kinetic characterization of RelA autoinhibition and activation by stalled ribosomes. Aim 2 will elucidate the structural mechanisms of RelA activation by stressed ribosomes primarily using cryo- EM. Aim 3 will dissect mechanisms of the dual enzyme SpoT, which possesses the hydrolase and weak synthetase activities and interacts with many molecular partners via unknown mechanisms. How the ribosome and other partners control opposing SpoT activities will be determined using biochemical and structural approaches. Completion of this project will expand the understanding of stringent response in bacteria, and may inform the development of new drugs to treat severe bacterial infections.

项目总结/摘要 作为儿童死亡的主要原因和成人死亡的主要原因，细菌感染仍然是最常见的疾病。 是全球健康的主要威胁。抗生素耐药性的出现速度超过了新抗生素的发展速度。 抗生素，并对传染病治疗构成严重挑战。了解细菌如何适应 感染期间的压力将刺激对新的治疗方法的探索。成为治疗目标， 这些机制必须在病原体中发挥关键的功能作用，但在哺乳动物有机体中则不然。 细菌的“严格反应”--细菌应激适应的主要调节因子--驱动细菌 致病机制和抗生素耐药性。严格的反应取决于RelA样蛋白， 调节第二信使分子pppGpp和ppGpp，其重新格式化细胞转录以适应 压力RelA样蛋白，如RelA和SpoT存在于大多数病原菌中。尽管许多细胞 和生化研究，RelA样蛋白的激活和严格反应的机制仍然存在 不太了解。 RelA样蛋白如何感知应激并催化pppGpp合成和水解尚不清楚。RelA是 一般认为是由脱酰基-tRNA激活，其在氨基酸剥夺反应中积累， 结合特定的mRNA密码子。奇怪的是，许多其他类型的压力激活严格的反应，我们的 初步的生化和结构研究表明，RelA和SpoT可以被核糖体激活， 不存在脱酰基-tRNA。拟议的研究将测试范式转变假设，即RelA样蛋白 通过核糖体停滞来感受许多压力，并且核糖体和其他激活剂诱导不同的压力， RelA和SpoT的构象变化来调节它们的活性。 该项目将利用生物化学和生物技术领域最近的方法创新和进展， 低温电子显微镜（cryo-EM）来剖析两种RelA样蛋白的分子机制。要求1 将通过识别以下功能来剖析RelA激活严格反应的生化基础： 调节结构域和动力学表征RelA的自抑制和激活停滞的核糖体。 目的2将阐明RelA激活应激核糖体的结构机制，主要是使用冷冻- EM.目的3剖析双酶SpoT的作用机制，该酶具有水解酶和弱 合成酶活性，并通过未知的机制与许多分子伴侣相互作用。核糖体是如何 和其他合作伙伴控制相反的SpoT活动将确定使用生化和结构 接近。该项目的完成将扩大对细菌严格反应的理解，并可能 为治疗严重细菌感染的新药开发提供信息。