Structural Biology of Multifunctional Bacterial Phosphatases

多功能细菌磷酸酶的结构生物学

• 批准号: 7631902
• 负责人: Matthew B Neiditch
• 金额: $ 39万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7631902
• 关键词: Anthrax disease; Anti-Bacterial Agents; Aspartic Acid; Bacillus (bacterium); Bacillus anthracis; Bacteria; Bacterial Genes; Bacterial Infections; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biological Assay; Cells; Chromosomes; Coma; Competence; Complex; Cytoplasmic Protein; DNA; Development; Drug Delivery Systems; Family; Gene Expression; Genetic; Goals; Growth; Human; In Vitro; Molecular; Organism; Overlapping Genes; Pathway interactions; Peptides; Pharmaceutical Preparations; Phenotype; Phosphoric Monoester Hydrolases; Plasmids; Protein Dephosphorylation; Proteins; Public Health; Repression; Research; Resistance; Roentgen Rays; Signal Transduction; Signal Transduction Pathway; Structure; System; Testing; Time; Virulence; Work; X-Ray Crystallography; bacterial genetics; design; genetic element; genetic regulatory protein; in vivo; pathogen; promoter; protein function; public health relevance; structural biology; transcription factor; vpr Genes

DESCRIPTION (provided by applicant): Rap proteins comprise a homologous family of cytoplasmic proteins that regulate bacterial gene expression via remarkably different mechanisms. Secreted signals, called Phr peptides, are imported into the cell where they bind to Rap proteins and repress their activities. The overall goal of our research is to determine how Rap-Phr signaling systems function mechanistically to regulate bacterial signal transduction. One subset of Rap proteins negatively regulates sporulation in B. subtilis by increasing the rate at which Spo0F, a central protein in the sporulation signal transduction pathway, catalyzes the dephosphorylation of a regulatory aspartic acid. Another subset of Rap proteins downregulates the development of genetic competence in B. subtilis by inhibiting ComA, the master transcriptional regulator of early competence gene expression, from binding to target DNA promoters. Additional B. subtilis Rap proteins that are not subjects of immediate study in this proposal regulate the mobility of genetic elements and antagonize the activity of transcription factors other than ComA. It is important from a public health standpoint to determine how Rap proteins regulate bacterial signal transduction because Rap proteins regulate virulence phenotypes in pathogenic organisms. For example, sporulation is repressed in Bacillus anthracis, the causative agent of the disease anthrax, by Rap proteins encoded on its chromosome and virulence plasmid, pX01. This repression is required for B. anthracis to become pathogenic vegetative cells in the infected host. How Rap proteins function mechanistically to regulate the diverse activities of their target proteins is not understood. Interestingly, Phr peptides are generated by an export maturation pathway from small proteins encoded by genes that overlap with the 3 end of the rap genes. Mature Phr pentapeptide molecules are imported into the cell where they bind to Rap proteins and inhibit their negative regulatory effects on gene expression. In Aim 1 we will determine how RapC negatively regulates genetic competence in B. subtilis by inhibiting the binding of ComA to target DNA promoters and also show how the secreted signal, PhrC, promotes genetic competence by inhibiting the interaction of RapC and ComA. In Aim 2 we will reveal how RapA inhibits B. subtilis sporulation by increasing the rate of Spo0F dephosphorylation and also determine how the secreted signal, PhrA, induces sporulation by inhibiting the interaction of RapA and Spo0F. The X-ray crystallographic, biochemical, and bacterial genetic studies proposed here will reveal, for the first time, how Rap proteins regulate the activities of their target proteins and how Phr peptides inhibit Rap protein function. Revealing the molecular mechanisms of Rap-Phr function will enable us to accomplish our long-term goal of designing antibacterial drugs that modulate bacterial signal transduction. PUBLIC HEALTH RELEVANCE Unique cellular proteins modulate the growth, proliferation, and virulence of bacteria, including common human pathogens. Bacterial infections are becoming increasingly difficult to treat, and an escalating threat to public health, as they acquire resistance to existing antibacterial drugs. The long-term goal of our work is to use biochemical, biophysical, and genetic approaches to elucidate the functions of bacterial regulatory proteins, and to design new classes of antibacterial drugs that target these proteins.

描述（由申请人提供）：Rap蛋白包含细胞质蛋白的同源家族，其通过显着不同的机制调节细菌基因表达。被称为 Phr 肽的分泌信号被输入细胞，与 Rap 蛋白结合并抑制其活性。我们研究的总体目标是确定 Rap-Phr 信号系统如何机械地发挥作用来调节细菌信号转导。 Rap 蛋白的一个子集通过增加 Spo0F（孢子形成信号转导途径的中心蛋白）催化调节性天冬氨酸去磷酸化的速率来负向调节枯草芽孢杆菌中的孢子形成。 Rap 蛋白的另一个子集通过抑制 ComA（早期能力基因表达的主要转录调节因子）与目标 DNA 启动子的结合，下调枯草芽孢杆菌遗传能力的发展。其他枯草芽孢杆菌 Rap 蛋白不是本提案中立即研究的主题，它们调节遗传元件的移动性并拮抗 ComA 以外的转录因子的活性。从公共卫生的角度来看，确定 Rap 蛋白如何调节细菌信号转导非常重要，因为 Rap 蛋白调节病原生物体的毒力表型。例如，炭疽杆菌（炭疽病的病原体）中的孢子形成受到其染色体和毒力质粒 pX01 上编码的 Rap 蛋白的抑制。这种抑制是炭疽杆菌在受感染宿主中成为致病性营养细胞所必需的。 Rap 蛋白如何通过机制调节其靶蛋白的多种活性尚不清楚。有趣的是，Phr 肽是通过输出成熟途径从与 rap 基因 3 端重叠的基因编码的小蛋白质产生的。成熟的 Phr 五肽分子被导入细胞，与 Rap 蛋白结合并抑制其对基因表达的负调控作用。在目标 1 中，我们将确定 RapC 如何通过抑制 ComA 与目标 DNA 启动子的结合来负向调节枯草芽孢杆菌的遗传能力，并展示分泌信号 PhrC 如何通过抑制 RapC 和 ComA 的相互作用来促进遗传能力。在目标 2 中，我们将揭示 RapA 如何通过增加 Spo0F 去磷酸化速率来抑制枯草芽孢杆菌孢子形成，并确定分泌信号 PhrA 如何通过抑制 RapA 和 Spo0F 的相互作用来诱导孢子形成。这里提出的X射线晶体学、生化和细菌遗传学研究将首次揭示Rap蛋白如何调节其靶蛋白的活性以及Phr肽如何抑制Rap蛋白功能。揭示 Rap-Phr 功能的分子机制将使我们能够实现设计调节细菌信号转导的抗菌药物的长期目标。公共卫生相关性 独特的细胞蛋白可调节细菌（包括常见的人类病原体）的生长、增殖和毒力。细菌感染变得越来越难以治疗，并且对公共健康的威胁不断升级，因为它们对现有抗菌药物产生了耐药性。我们工作的长期目标是利用生物化学、生物物理和遗传学方法来阐明细菌调节蛋白的功能，并设计针对这些蛋白的新型抗菌药物。