Regulation of eukaryotic histidine kinases

真核组氨酸激酶的调控

• 批准号: 6675004
• 负责人: Robert J Deschenes
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6675004
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; biochemistry; biological signal transduction; electron microscopy; eukaryote; fungal genetics; fungal proteins; gel electrophoresis; histidine; nuclear magnetic resonance spectroscopy; osmotic pressure; polymerase chain reaction; protein kinase; protein structure; protein structure function; transcription factor

A basic requirement of all cells is the ability to sense and respond to changes in the environment. Osmolarity is one of the most basic conditions to which cells must respond. Despite the ubiquitous nature of osmosensing systems, the molecular mechanism by which osmotic pressure is sensed is largely unknown. Studies involving microbial model systems have played an important role in identifying the sensors and signal transduction pathways that respond to changes in osmolarity. We plan to use the yeast osmotic stress sensor, Slnlp, as a paradigm for eukaryotic osmosensors. Changes in osmotic conditions regulate Slnlp causing changes in phosphate flux between the individual proteins comprising the two-component signaling system. The fundamental question addressed by this proposal is how Slnlp activity is regulated in response to changes in osmolarity. Using computational, genetic, and biochemical techniques, we have identified a coiled-coil dimerization domain that plays a key role in mediating the stimulus-activation step. It is located in the linker region between the membrane and the kinase domain. The specific objective of this application is to perform a detailed structure-function analysis of the coiled coil (CC) region of the yeast Slnlp osmosensor to test the hypothesis that the unusual composition of the HK CC contributes to the regulation of the HK family of sensor kinases. The specific aims of the proposal are to (1) Genetically dissect the Sin1 CC domain, (2) Determine the structure of the CC domain and CC mutants, and (3) Develop membrane-based assays for Sin1 function. The elucidation of the unique structural and mechanistic features of the two-component type coiled-coil domain in Slnlp will serve as a model for this class of signaling molecules and may lead to the development of histidine kinase inhibitors for antifungal therapy.

所有细胞的基本要求是感知和响应环境变化的能力。 渗透性是细胞必须响应的最基本条件之一。尽管渗透压系统无处不在，但感觉到渗透压的分子机制在很大程度上是未知的。涉及微生物模型系统的研究在识别传感器和信号转导途径方面发挥了重要作用，这些传感器和信号转导途径响应渗透压变化。我们计划使用酵母渗透应力传感器SLNLP作为真核渗透器的范式。渗透条件的变化调节SLNLP，导致构成两个组件信号系统的单个蛋白质之间的磷酸盐通量变化。该提议解决的基本问题是如何在SLNLP活动中进行调节 对渗透压变化的响应。使用计算，遗传和生化技术，我们确定了一个盘绕螺旋二聚体域，该结构域在介导刺激激活步骤中起着关键作用。它位于膜和激酶结构域之间的接头区域。该应用的具体目标是对酵母SLNLP Osmosensor的盘绕线圈（CC）区域进行详细的结构 - 功能分析，以检验以下假设：HK CC的异常组成有助于调节传感器激酶的HK家族。该提案的具体目的是（1）基因剖析SIN1 CC结构域，（2）确定CC域和CC突变体的结构，以及（3）开发基于膜的SIN1功能测定法。阐明SLNLP中两种组件类型的盘绕螺旋结构域的独特结构和机械特征将作为此类信号分子的模型，并可能导致抗真菌剂的组氨酸激酶抑制剂的发展 治疗。