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Molecular Mechanisms of Phosphate Signaling in E. coli

大肠杆菌中磷酸盐信号传导的分子机制

基本信息

批准号：
8035126
负责人：
WILLIAM R MCCLEARY
金额：
$ 28.66万
依托单位：
BRIGHAM YOUNG UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8035126
关键词：
Address Affinity Amino Acids Bacteria Bacterial Infections Basic Science Biochemical Biological Cell physiology Cells Chimeric Proteins Co-Immunoprecipitations Complex Development Disease Drug Design Environment Escherichia coli Eukaryota Event Gene Expression Genes Genetic Heart Human Immune system Inorganic Phosphate Transporter Ligand Binding Mediating Membrane Molecular Molecular Biology Molecular Conformation Mutate Mutation Pathway interactions Pharmaceutical Preparations Phosphorylation Process Protein Dephosphorylation Proteins Public Health Recording of previous events Regulation Research Research Proposals Role Sensory Signal Pathway Signal Transduction Signal Transduction Pathway Signaling Protein Site-Directed Mutagenesis System Transport Process Work antimicrobial drug base combat genetic analysis inorganic phosphate mutant pathogen protein function protein protein interaction protein-histidine kinase research study response sensor sensor histidine kinase transmission process yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): The objective of the proposed research is to understand how information concerning environmental phosphate levels is transmitted through the phosphate signal transduction pathway to control gene expression in Escherichia coli. At the heart of this pathway are the response regulator PhoB and the histidine kinase PhoR. A great deal of work has already been accomplished elucidating the mechanisms by which these two proteins function. The focus of this proposal is to understand how two auxiliary proteins, PstSCAB2 and PhoU, are involved in the sensing of phosphate and the control of this signaling pathway. Given the history of research into phosphate regulation, it is somewhat surprising that fundamental questions still exist as to how cells sense phosphate and how that information is transmitted within a cell. PstSCAB2 is a phosphate transporter that also functions as the sensor for the system. PhoU controls the activity of PstSCAB2 and is required for the transmission of the signal from PstSCAB2 to PhoR. Our first hypothesis is that the PstSCAB2 transporter transduces information about environmental phosphate levels through conformational changes that are inherent to the transport process. Our second hypothesis is that PhoU transmits this information by specifically interacting with a particular PstSCAB2 conformation and mediates protein/protein interactions with PhoR. We plan on addressing these hypotheses through both genetic and biochemical approaches. Mutant versions of the transporter will be isolated that "lock" it into various conformations. We will use these versions of the transporter to trap complexes with other proteins using co-elution and co-immunoprecipitation experiments and as well as bacterial two-hybrid analysis. These approaches should prove to be complementary to one another and will provide the greatest opportunity to observe protein/protein interactions within the proposed signaling complex. In the analysis of the role of PhoU, constitutive signaling mutants will be isolated in the absence of the transporter and studied for interactions with PhoR. In addition, highly conserved amino acid residues of PhoU, which must be important to its function, will be mutated and studied for function and cellular localization. PhoU-GFP fusions will also be constructed and used to study localization. The proposed work is important because these signaling proteins are essential for bacteria to survive changing environments - including the human immune system. This feature combined with their absence in higher eukaryotes, makes this signaling pathway a targets for the development of new antimicrobial drugs. An increased understanding of these signal transduction proteins may assist in the rational design of drugs to combat pathogens. PUBLIC HEALTH RELEVANCE: This project, which focuses on determining how bacterial cells sense phosphate to control various survival genes, is relevant to public health because it provides foundational basic research into a fundamental process of bacteria. The phosphate sensory response pathway is essential to many pathogens in their ability to cause disease. Because of this research, strategies and drugs may be developed that will limit a pathogens survival, thereby helping to combat various bacterial infections.

描述（由申请人提供）：拟议研究的目的是了解有关环境磷酸盐水平的信息如何通过磷酸盐信号转导途径传递，以控制大肠杆菌中的基因表达。该途径的核心是反应调节剂PhoB和组氨酸激酶PhoR。已经完成了大量的工作来阐明这两种蛋白质的功能机制。该提案的重点是了解两种辅助蛋白PstSCAB 2和PhoU如何参与磷酸盐的传感和该信号通路的控制。鉴于磷酸盐调节研究的历史，令人惊讶的是，细胞如何感知磷酸盐以及这些信息如何在细胞内传递的基本问题仍然存在。PstSCAB 2是一种磷酸盐转运蛋白，也可用作系统的传感器。PhoU控制PstSCAB 2的活性，并且是将信号从PstSCAB 2传输到PhoR所必需的。我们的第一个假设是，PstSCAB 2转运蛋白通过运输过程中固有的构象变化转导有关环境磷酸盐水平的信息。我们的第二个假设是，PhoU通过与特定的PstSCAB 2构象特异性相互作用来传递这些信息，并介导与PhoR的蛋白质/蛋白质相互作用。我们计划通过遗传和生物化学方法来解决这些假设。突变版本的转运蛋白将被隔离，将其“锁定”为各种构象。我们将使用这些版本的转运蛋白捕获复合物与其他蛋白质使用共洗脱和免疫共沉淀实验，以及细菌双杂交分析。这些方法应该被证明是相互补充的，并将提供最大的机会，观察蛋白质/蛋白质相互作用的建议信号复合物。在分析PhoU的作用时，将在不存在转运蛋白的情况下分离组成型信号转导突变体，并研究其与PhoR的相互作用。此外，高度保守的氨基酸残基的PhoU，这必须是重要的，它的功能，将被突变和功能和细胞定位的研究。还将构建PhoU-GFP融合体并用于研究定位。这项工作很重要，因为这些信号蛋白是细菌在不断变化的环境中生存所必需的，包括人类免疫系统。这一特征与它们在高等真核生物中的缺失相结合，使得这种信号通路成为开发新的抗微生物药物的靶点。对这些信号转导蛋白的进一步了解可能有助于合理设计药物来对抗病原体。公共卫生相关性：该项目的重点是确定细菌细胞如何感知磷酸盐以控制各种生存基因，与公共卫生有关，因为它为细菌的基本过程提供了基础性的基础研究。磷酸盐感觉反应途径对于许多病原体致病能力至关重要。由于这项研究，可能会开发出限制病原体存活的策略和药物，从而有助于对抗各种细菌感染。