RIG: Regulated Intramembrane Proteolytic Activation of Membrane-anchored Transcription Factors in Anabaena

RIG：鱼腥藻膜锚定转录因子的调节膜内蛋白水解激活

• 批准号: 0914691
• 负责人: Ruanbao Zhou
• 金额: $ 17.2万
• 依托单位: South Dakota State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-18 至 2012-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0914691&HistoricalAwards=false
• 关键词: RIG; Regulated; Intramembrane; Proteolytic; Activation

Intellectual Merit The regulation of gene transcription is one of the most important mechanisms used by cells to respond to alterations in their internal or external environment. To ensure a rapid response, transcriptional regulators are often synthesized and stored in a dormant form, becoming active only in response to a specific signal. Much of the biological interest in transcriptional responses focuses on how the activity of transcription factors is regulated. Sequestration of transcription factors away from their DNA targets, by temporary attachment to cellular membranes, is emerging as an elegant mechanism to regulate their activities. Several membrane-anchored transcription factors (MTFs) have been experimentally identified whose access to the transcriptional machinery is regulated by proteolytic cleavage from the membrane. This process is broadly referred to as regulated intramembrane proteolysis (RIP). RIP is a newly discovered mechanism that controls many important signaling pathways conserved from bacteria to humans. The discovery of an essential role for RIP in biologically relevant processes makes it important to understand the full gamut of RIP function. However, RIP is little understood and so far there has been no systematic study of RIP in any organism. This project is for a genome-wide study of RIP in Anabaena variabilis 29413, a multicellular cyanobacterium, capable of simultaneously carrying out agriculturally relevant O2-producing photosynthesis and O2-labile N2-fixation, as well as bio-solar H2 gas production. In addition, its vegetative cells can differentiate into three other types of cells in response to environmental changes, which presents a rare opportunity to investigate gene regulation of several differentiation processes in a single bacterium. Given its unique capabilities, A. variabilis provides a very attractive experimental system to study fundamental biological problems, especially for agriculture and bio-energy related problems. An analysis of the genomic sequence of A. variabilis identified 19 putative RIP genes that encode 5 RIP proteases and 14 MTFs. Literature surveys imply that some orthologs of the putative RIP genes may be involved in regulation of stress responses, chloroplast development, cellular differentiation, cell-cell communication, and photosynthesis, as well as aerobic N2-fixation. Therefore, the 19 putative RIP genes in A. variabilis may play important biological roles. The aims of the project are 1) to obtain clues for the cellular functions of 19 putative RIP genes by inactivating these genes; 2) to determine the expression patterns of these genes for further definition of their functions during A. variabilis growth and development, and in response to various stress conditions; 3) to determine the proteolytic activities of 5 putative RIP proteases in a reconstituted E. coli system; and 4) to identify specific MTF/RIP protease pairs. Broader Impact The PI has a history of mentoring students and in this project will broaden participation through outreach activities in an established collaboration at Michigan State University with a minority female professor who participates in the Charles Drew program. The Charles Drew Program is a retention and academic enhancement program for high-achieving undergraduate students and graduates from underrepresented minority groups. The PI will mentor students in the program, provide career development guidance to the participants, and serve as a research advisor to interested students. In this project the PI will provide hands-on research experiences to four undergraduates.

基因转录调控是细胞应对内外环境变化的最重要机制之一。为了确保快速反应，转录调节因子通常被合成并以休眠形式存储，仅在响应特定信号时才变得活跃。许多生物学对转录反应的兴趣集中在如何调节转录因子的活性上。通过暂时附着在细胞膜上，将转录因子从它们的DNA目标中隔离出来，这是一种调节转录因子活动的优雅机制。几个膜锚定转录因子（MTFs）已经被实验确定，其进入转录机制是由膜的蛋白水解裂解调节的。这一过程被广泛地称为调节膜内蛋白水解（RIP）。RIP是一种新发现的机制，它控制着从细菌到人类的许多重要信号通路。RIP在生物学相关过程中的重要作用的发现使得了解RIP功能的全部范围变得重要。然而，人们对RIP知之甚少，迄今为止还没有对任何生物体的RIP进行系统的研究。该项目是对Anabaena variabilis 29413的RIP进行全基因组研究，Anabaena variabilis 29413是一种多细胞蓝藻，能够同时进行与农业相关的产生o2的光合作用和不稳定的o2固定，以及生物太阳能氢气生产。此外，它的营养细胞可以根据环境变化分化成三种其他类型的细胞，这为研究单个细菌中几种分化过程的基因调控提供了难得的机会。由于其独特的能力，变异芽孢杆菌提供了一个非常有吸引力的实验系统来研究基础生物学问题，特别是与农业和生物能源相关的问题。对变异性A. variabilis基因组序列的分析鉴定出19个推测的RIP基因，这些基因编码5个RIP蛋白酶和14个mtf。文献调查表明，一些推测的RIP基因的同源物可能参与调控应激反应、叶绿体发育、细胞分化、细胞间通讯、光合作用以及需氧氮固定。因此，19个推测的RIP基因可能在变异性假单胞菌中发挥重要的生物学作用。该项目的目的是：1)通过灭活19个推测的RIP基因来获得细胞功能的线索；2)确定这些基因的表达模式，以进一步明确其在变异性拟南芥生长发育过程中以及在不同胁迫条件下的功能；3)在重组大肠杆菌体系中测定5种推测的RIP蛋白酶的蛋白水解活性；4)鉴定特异性MTF/RIP蛋白酶对。更广泛的影响PI有指导学生的历史，在这个项目中，将通过与密歇根州立大学一位参加查尔斯·德鲁项目的少数族裔女教授的既定合作，开展外展活动，扩大参与范围。查尔斯·德鲁计划是一个保留和学术提升计划，为来自代表性不足的少数群体的优秀本科生和毕业生提供服务。PI将在项目中指导学生，为参与者提供职业发展指导，并为感兴趣的学生担任研究顾问。在这个项目中，PI将为四名本科生提供实践研究经验。