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蛋白包括一个同源的细胞质蛋白家族，该家族通过明显不同的机制来调节细菌基因的表达。分泌的信号（称为PHR肽）被进口到与RAP蛋白结合并抑制其活性的细胞中。我们研究的总体目标是确定RAP-PHR信号系统如何机械地调节细菌信号转导。 RAP蛋白的一个子集通过增加SPO0F的速率（Spo0F）（一种孢子型信号转导途径中的中心蛋白）对枯草芽孢杆菌的孢子产生负调节，从而催化了调节性天冬氨酸的去磷酸化。 RAP蛋白的另一个子集通过抑制早期能力基因表达的主要转录调节剂的昏迷来下调枯草芽孢杆菌中遗传能力的发展，从与靶DNA启动子结合。在该提案中不是立即研究的主体的其他枯草芽孢杆菌RAP蛋白调节遗传元素的迁移率，并与昏迷以外的转录因子的活性拮抗。从公共卫生的角度来看，重要的是说RAP蛋白如何调节细菌信号转导，因为RAP蛋白调节致病生物的毒力表型。例如，在其染色体和毒力质粒PX01上编码的RAP蛋白PRAP蛋白P X01在炭疽芽孢杆菌（炭疽芽孢杆菌）中受到了孢子形成。 B.炭疽菌成为受感染宿主的致病植物细胞需要这种抑制。尚不了解RAP蛋白如何机械地调节其靶蛋白的各种活性。有趣的是，PHR肽是由从与RAP基因3端重叠的基因编码的小蛋白的出口成熟途径产生的。成熟的PHR五肽分子被进口到与RAP蛋白结合的细胞中，并抑制其对基因表达的负调节作用。在AIM 1中，我们将通过抑制昏迷与靶向DNA启动子的结合来确定RAPC如何负面调节枯草芽孢杆菌中的遗传能力，并显示分泌的信号如何通过抑制RAPC和昏迷的相互作用来促进遗传能力。在AIM 2中，我们将通过增加SPO0F去磷酸化的速率来揭示RAPA如何抑制枯草芽孢杆菌的孢子形成，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。此处提出的X射线晶体学，生化和细菌遗传研究将首次揭示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。细菌感染变得越来越难以治疗，并且对公共卫生的威胁升级，因为它们对现有的抗菌药物有抵抗力。我们工作的长期目标是使用生化，生物物理和遗传方法来阐明细菌调节蛋白的功能，并设计针对这些蛋白质的新型抗菌药物。