Structural Biology of Multifunctional Bacterial Phosphatases

多功能细菌磷酸酶的结构生物学

• 批准号: 8117171
• 负责人: Matthew B Neiditch
• 金额: $ 38.22万
• 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/8117171
• 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; Health; 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; 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(申请人提供)：RAP蛋白包括一个同源的细胞质蛋白家族，通过显著不同的机制调节细菌的基因表达。被称为Phr肽的分泌信号被输入细胞，在那里它们与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的信号转导。在枯草杆菌中，Rap蛋白的一个亚群通过增加产孢子信号转导途径中的中心蛋白Spo0F催化调节天冬氨酸的去磷酸化的速率来负向调节产孢子。另一组Rap蛋白通过抑制ComA(早期能力基因表达的主要转录调节因子)与靶DNA启动子结合来下调枯草杆菌遗传能力的发展。另外，枯草杆菌Rap蛋白不是本建议中立即研究的对象，它们调节遗传元件的流动性，并拮抗除昏迷之外的转录因子的活性。从公共卫生的角度来看，确定Rap蛋白如何调节细菌的信号转导是很重要的，因为Rap蛋白调节病原生物体的毒力表型。例如，在炭疽病的病原体炭疽杆菌中，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营养细胞所必需的。Rap蛋白如何机械地发挥作用来调节其目标蛋白的不同活性尚不清楚。有趣的是，Phr多肽是由与RAP基因3端重叠的基因编码的小蛋白通过出口成熟途径产生的。成熟的Phr五肽分子被输入细胞，在那里它们与Rap蛋白结合，并抑制它们对基因表达的负面调控作用。在目标1中，我们将确定RAPC如何通过抑制COMA与靶DNA启动子的结合来负面调节枯草杆菌的遗传能力，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去磷酸化来抑制枯草杆菌产孢量，并确定分泌信号PhrA如何通过抑制RapA和Spo0F的相互作用来诱导产孢量。这里提出的X射线结晶学、生化和细菌遗传学研究将首次揭示Rap蛋白如何调节其目标蛋白的活性，以及Phr肽如何抑制Rap蛋白的功能。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。与公共健康相关独特的细胞蛋白调节细菌的生长、增殖和毒力，包括常见的人类病原体。细菌感染正变得越来越难以治疗，对公共健康的威胁也在不断升级，因为它们对现有的抗菌药物产生了抗药性。我们工作的长期目标是使用生化、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。