UWY COBRE: MECHANISMS OF HYPOXIA SENSING FROM RHODOBACTER TO HUMANS

UWY COBRE：红细菌对人类的缺氧感知机制

• 批准号: 7381216
• 负责人: Mark Gomelsky
• 金额: $ 30.78万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381216
• 关键词: gene expression; hypoxia

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sensing and responding to hypoxia are of critical importance to most organisms. Hypoxia is a pathophysiological component of the major causes of mortality in Western societies: heart attack, stroke, and cancer. Many molecular mechanisms of hypoxia sensing are conserved from unicellular prokaryotes to humans. Furthermore, proteins mediating hypoxic responses contain similar structural modules, a representative example being the PAS domains. One goal of this project is to elucidate a mechanism of hypoxia sensing in a mitochondria lineage bacterium Rhodobacter sphaeroides that involves a PAS domain-containing transcriptional repressor PpsR. Elucidation of the molecular mechanism of hypoxia sensing by PpsR as well as characterization of the components involved in the signal transduction cascade could shed light on the mechanisms of hypoxia dependent gene expression in mammals. We studied the roles of various domains of PpsR by using two-hybrid screens, domain-domain interactions in vitr o and mutant analysis. We found that the PAS domains of PpsR are involved in protein-protein interactions, i.e. similar to their role in the eukaryotes. It is the first demonstration of this function of the PAS domains in prokaryotes. To elucidate the hypoxia dependent signal transduction cascade, we are searching for proteins that might interact with PpsR and constructing mutations in PpsR that might affect its redox sensitivity. We reached an Agreement with Affymetrix, Inc. (Santa Clara, CA) for production of the R. sphaeroides GeneChips that will be used for whole genome assessment of hypoxia dependent gene expression. We optimized conditions for sample preparation and performed initial experiments on gene expression profiling in mice using mouse GeneChips. We are in a position to start characterizing global gene expression in mice experiencing artificially induced ischemia.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。对大多数生物来说，感知和响应低氧是至关重要的。在西方社会，缺氧是心脏病发作、中风和癌症等主要死亡原因的病理生理成分。从单细胞原核生物到人类，许多缺氧感知的分子机制都是保守的。此外，介导低氧反应的蛋白质含有类似的结构模块，一个典型的例子是PAS结构域。该项目的一个目标是阐明线粒体谱系细菌球形红假单胞菌中的低氧感知机制，该机制涉及含有PAS结构域的转录抑制因子PpsR。阐明PpsR感知低氧的分子机制以及对信号转导级联反应的研究有助于揭示哺乳动物低氧依赖基因表达的机制。我们通过双杂交筛选、结构域-结构域相互作用和突变分析研究了PpsR的各个结构域的作用。我们发现PpsR的PAS结构域参与蛋白质-蛋白质的相互作用，即与它们在真核生物中的作用相似。这是原核生物中PAS结构域这一功能的首次证明。为了阐明低氧依赖的信号转导级联反应，我们正在寻找可能与PpsR相互作用的蛋白质，并构建可能影响其氧化还原敏感性的PpsR突变。我们与Affymetrix，Inc.(圣克拉拉，加利福尼亚州)达成了一项协议，生产球形R.sphaeroidesGeneChips，用于低氧依赖基因表达的全基因组评估。我们优化了样品制备条件，并使用小鼠GeneChips对小鼠的基因表达谱进行了初步实验。我们能够开始描述经历人工诱导的缺血小鼠的整体基因表达。