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Structural Biology of Multifunctional Bacterial Phosphatases

多功能细菌磷酸酶的结构生物学

基本信息

批准号：
8711660
负责人：
Matthew B Neiditch
金额：
$ 12.55万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8711660
关键词：
Structural Biology Multifunctional Bacterial Phosphatases

项目摘要

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.

描述（由申请人提供）：Rap蛋白包括通过显著不同的机制调节细菌基因表达的同源细胞质蛋白家族。被称为Phr肽的分泌信号被导入细胞，在那里它们与Rap蛋白结合并抑制它们的活性。我们研究的总体目标是确定Rap-Phr信号系统如何机制地调节细菌信号转导。Rap蛋白的一个子集负调节B中的孢子形成。通过增加Spo 0 F（孢子形成信号转导途径中的中心蛋白）催化调节性天冬氨酸的去磷酸化的速率来使枯草芽孢杆菌的细胞凋亡。Rap蛋白的另一个子集下调B中遗传能力的发展。通过抑制早期感受态基因表达的主要转录调节因子ComA与靶DNA启动子的结合，附加B。枯草杆菌Rap蛋白不是本建议中直接研究的对象，其调节遗传元件的移动性并拮抗除ComA之外的转录因子的活性。从公共卫生的角度来看，确定Rap蛋白如何调节细菌信号转导是重要的，因为Rap蛋白调节致病生物体中的毒力表型。例如，在炭疽芽孢杆菌（炭疽病的病原体）中，孢子形成被其染色体上编码的Rap蛋白和毒力质粒pX 01抑制。这种抑制是B所必需的。炭疽菌在被感染的宿主中变成致病性营养细胞。Rap蛋白如何机械地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，在那里它们与Rap蛋白结合并抑制它们对基因表达的负调节作用。在目标1中，我们将确定RapC如何负调控B中的遗传能力。通过抑制ComA与靶DNA启动子的结合，对枯草芽孢杆菌进行了研究，还显示了分泌信号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如何抑制B。通过增加Spo 0 F去磷酸化的速率来确定枯草芽孢杆菌孢子形成，并且还确定分泌的信号PhrA如何通过抑制RapA和Spo 0 F的相互作用来诱导孢子形成。本文提出的X射线晶体学、生物化学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。