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

基因转录调控是细胞对其内外环境变化作出反应的最重要的机制之一。为了确保快速反应，转录调节因子通常被合成并以休眠的形式存储，只有在对特定信号做出反应时才变得活跃。对转录反应的生物学兴趣主要集中在转录因子的活性是如何被调节的。通过暂时附着在细胞膜上，将转录因子从其DNA靶标中隔离出来，正在成为一种调节其活性的优雅机制。一些膜锚定转录因子(MTF)已经被实验确定，它们进入转录机制的途径是由膜上的蛋白水解性切割来调节的。这个过程被广泛地称为调控的膜内蛋白分解(RIP)。RIP是一种新发现的机制，它控制着许多从细菌到人类保守的重要信号通路。RIP在生物学相关过程中的重要作用的发现使得理解RIP功能的全域变得重要。然而，人们对RIP知之甚少，到目前为止，还没有对任何生物体中的RIP进行系统的研究。这个项目是为了在全基因组范围内研究可变鱼腥藻29413中的RIP，这是一种多细胞蓝藻，能够同时进行与农业相关的产生O2的光合作用和不稳定O2的固氮，以及生物太阳能产生H2气体。此外，它的营养细胞可以分化为其他三种类型的细胞来响应环境的变化，这为研究单一细菌中几个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，变异藻类提供了一个非常有吸引力的实验系统来研究基本的生物学问题，特别是与农业和生物能源相关的问题。通过对变异链霉菌基因组序列的分析，确定了19个可能的RIP基因，编码5个RIP酶和14个MTF。文献研究表明，RIP基因的某些同源基因可能参与胁迫反应、叶绿体发育、细胞分化、细胞间通讯、光合作用以及好氧固氮等的调控。因此，该19个可能的RIP基因可能在变种拟青霉中发挥重要的生物学作用。该项目的目标是：1)通过失活19个RIP基因来获得这些基因的细胞功能线索；2)确定这些基因的表达模式，以便进一步确定它们在变形虫生长发育过程中的功能，以及对各种胁迫条件的响应；3)在重组的大肠杆菌系统中确定5个可能的RIP酶的蛋白水解酶活性；以及4)鉴定特定的MTF/RIP蛋白酶对。更广泛的影响国际和平协会有指导学生的历史，在这个项目中，将通过在密歇根州立大学与一名参与查尔斯·德鲁项目的少数族裔女教授的既定合作开展外联活动，扩大参与范围。查尔斯·德鲁计划是为成绩优异的本科生和来自未被充分代表的少数族裔群体的毕业生提供的留住和学业提升计划。PI将指导项目中的学生，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体验。

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.