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.

项目摘要/摘要 作为儿童死亡率的主要原因和成人死亡率的主要原因，细菌感染仍然是 全球卫生的主要威胁。抗生素耐药性的出现超过了新的发展 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寻找新型治疗方法。成为治疗靶标的 这些机制必须在病原体中起关键的功能作用，而在哺乳动物生物中则不得不发挥作用。 细菌“严格反应” - 细菌应力适应的主要调节剂 - 驱动细菌 发病机理和抗生素耐药性。严格的响应取决于产生的类似Rela的蛋白质 调节第二个信使分子PPPGPP和PPGPP，它们重新格式化转录以适应 压力。在大多数致病细菌中存在类似Rela样蛋白，例如Rela和斑点。尽管有许多细胞 和生化研究，相对样蛋白和严格反应的激活机制仍然存在 理解不佳。 Rela样蛋白如何感应应力并催化PPPGPP的合成和水解尚不清楚。 Rela是 通常认为是被脱酰基trnas激活的，这些脱酰基因氨基酸的剥夺而积累 结合特定的mRNA密码子。矛盾的是，许多其他类型的压力激活了严格的反应，我们的 初步的生化和结构研究表明，Rela和斑点可以被核糖体激活 缺乏脱酰基-TRNA。拟议的研究将测试范式转移假设，即相反的蛋白 通过核糖体停滞来感知许多压力，核糖体和其他激活剂会引起不同的压力 Rela和Lock的构象变化以调节其活动。 该项目将利用最新的方法论创新和生物化学的进步和 低温电子显微镜（Cryo-EM），以剖析两种类似蛋白的分子机制。目标1 将通过识别RELA的严格响应激活的生化基础来识别严格的响应激活 调节结构域以及通过停滞核糖体对Rela自身抑制和激活的动力学表征。 AIM 2将通过压力核糖体阐明重复激活的结构机制，主要使用冷冻 Em。 AIM 3将剖析具有水解酶和弱的双酶斑点的机制 合成酶活性并通过未知机制与许多分子伴侣相互作用。核糖体如何 其他合作伙伴控制相反的现场活动将使用生化和结构性确定 方法。该项目的完成将扩大对细菌中严格反应的理解，并可能 告知新药的开发以治疗严重的细菌感染。